Category Archives: Cloning

Patrick Wright, WVEC 8:53 AM. MST February 01, 2017

GREENSBORO, N.C. -- Most Facebook friend requests come from actual friends, but some are from anonymous people with ulterior motives. And, if you fall for their tricks, it could cost you.

Some hackers are using a tactic called "Facebook Cloning." They steal your Facebook name, add your friends and use your photos to clone your account. Then, they use the fake account to approach your friends and family online.

"Maybe theyre trying to get you to send them money," said Danielle Hatfield, owner of Experience Farm. "However, other scammers are trying to do something a little more nefarious, and thats steal your identity."

Hatfield says the clonersmight even check your statuses to learn to mimic your style of communication.

"When they finally get around to the scam of maybe asking for money, your friends and family will fall for it.," Hatfield said.

13News Now sister stationWFMY News 2's Patrick Wright's great aunt fell victim to Facebook cloning.

"I get upset because this is about the third time this has happened," Yvonne Allen said.

A fake account, made to look like Allen's, reached out to Patrick on Facebook Messenger. The user told him they'd received a $50 million grant from the government and wanted to share the news of how others could get their own.

"I didnt receive anything! If they want to send me $50 million, Ill take it," Allen laughed.

Hatfield says, if you come across an account you aren't sure is real, just search it on Facebook to see if you're already friends with that person. If they send you a questionable post or link, give them a call or text message and ask if it's really them. If the account is fake, report it to Facebook immediately.

If your account gets cloned, Hatfield says you should change your password, warn others, and then check your privacy settings to make sure only friends can view your profile.

( 2017 WFMY)

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Beware Of 'Facebook Cloning' | 9news.com - 9NEWS.com

ScienceBlog.com (blog)

Crustacean Cloning The Poetry of Science ScienceBlog.com (blog) A typical example of a Lybia crab holding a sea anemone in each claw (Photo Credit: Yisrael Schnytzer). This is a Rondelet, inspired by recent research that ... and more »

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Crustacean Cloning The Poetry of Science - ScienceBlog.com (blog)

JoVE Science Education Basic Biology Basic Methods in Cellular and Molecular Biology Molecular Cloning

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Molecular cloning is a set of techniques used to insert recombinant DNA from a prokaryotic or eukaryotic source into a replicating vehicle such as plasmids or viral vectors. Cloning refers to making numerous copies of a DNA fragment of interest, such as a gene. In this video you will learn about the different steps of molecular cloning, how to set up the procedure, and different applications of this technique.

At least two important DNA molecules are required before cloning begins. First, and most importantly, you need the DNA fragment you are going to clone, otherwise known as the insert. It can come from a prokaryote, eukaryote, an extinct organism, or it can be created artificially in the laboratory. By using molecular cloning we can learn more about the function of a particular gene.

Second, you need a vector. A vector is plasmid DNA used as a tool in molecular biology to make more copies of or produce a protein from a certain gene. Plasmids are an example of a vector, and are circular, extra chromosomal, DNA that is replicated by bacteria.

A plasmid typically has a multiple cloning site or MCS, this area contains recognition sites for different restriction endonucleases also known as restriction enzymes. Different inserts can be incorporated into the plasmid by a technique called ligation. The plasmid vector also contains an origin of replication, which allows it to be replicated in bacteria. In addition, the plasmid has an antibiotic gene. If bacteria incorporate the plasmid, it will survive in media that contains the antibiotic. This allows for the selection of bacteria that have been successfully transformed.

The insert and vector are cloned into a host cell organism, the most common used in molecular cloning is E. coli. E. coli grows rapidly, is widely available and has numerous different cloning vectors commercially produced. Eukaryotes, like, yeast can also be used as host organisms for vectors.

The first step of the general molecular cloning procedure is to obtain the desired insert, which can be derived from DNA or mRNA from any cell type. The optimal vector and its host organism are then chosen based they type of insert and what will ultimately be done with it. A polymerase chain reaction, or PCR based method is often used to replicate the insert.

Then by using a series of enzymatic reactions, the insert and digest are joined together and introduced into the host organism for mass replication. Replicated vectors are purified from bacteria, and following restriction digestion, analyzed on a gel. Gel-purified fragments are later sent for sequencing to verify that the inset is the desired DNA fragment.

Lets have a little more detailed look at how molecular cloning is conducted. Before beginning, you will want to plan out your cloning strategy, prior to making any cloning attempt at the bench. For example, any given plasmid vector, will provide you with a finite number of restriction sites to incorporate the insert via the multiple cloning site. Youll need to choose restriction sites that are not found in your insert so that you do not cleave it. You might be left with a situation where you are forced to join a blunt end fragment with one that has an overhang. If so, then using the klenow fragment to set up a blunt end ligation might be your only option to get the insert into your desired vector. Understanding the various molecular cloning tools at your disposal, as well as coming up with a careful strategy before you begin cloning can be an immense time saver.

The source of DNA for molecular cloning can be isolated from almost any type of cell or tissue sample through simple extraction techniques. Once isolated, PCR can be used to amplify the insert.

Once the insert is amplified both it and the vector are digested by restriction enzymes, also known as restriction endonucleases.

Once digested, the insert and vector can be run on a gel and purified by a process called gel purification. With respect to the vector, this step will help to purify linearized plasmid from uncut plasmid, which tends to appear as a high molecular weight smear on a gel.

After gel purifying the digests, the insert is ligated or joined to the plasmid, via an enzyme called DNA ligase.

Generally speaking, it is always a good idea to set up ligations, so that the ratio of insert to vector is 3 to 1, which ensures that only a small amount of vector will self-ligate. Once the ligation has been set up on ice, it is incubated anywhere from 14-25C from 1 hr to overnight.

Next, transformation is performed to introduce the plasmid vector into the host that will replicate it.

Following transformation bacteria are plated on agar plates with antibiotic and incubated overnight at 37C. Because the plasimid contains an antibiotic resistance gene, successful transformation will produce bacterial colonies when grown on agar plates in presence of antibiotics. Individual colonies can then be picked from the transformed plate, placed into liquid growth media in numbered tubes, and put into a shaking incubator for expansion. A small volume of liquid culture is added to a numbered agar plate, while the rest of the culture moves on to plasmid purification. The numbering scheme that denotes the identity of bacterial colonies from which the plasmids will eventually be purified is maintained throughout the plasmid purification process.

A sample of purified plasmid is then cut with restriction enzymes. The digest is then loaded and run on the gel in order to check for the presence of insert, which will verify that the bacterial colony was transformed with a plasmid containing an insert and not self-ligated plasmid. Bacteria verified to have been transformed with an insert-containing plasmid, are expanded for further plasmid purification. Sequencing is used performed as a final verification step to confirm that your gene of interest has been cloned.

Molecular cloning can be used for a near limitless number of applications. For instance, when an mRNA template is reverse transcribed to form cDNA, or complementary DNA, by an enzyme called reverse transcriptase and then PCR is used to amplify the cDNA, molecular cloning can be used to create a cDNA library a library of all of the genes expressed by a given cell type.

Molecular cloning can also be employed to take a series of genes, or gene cluster from one bacterial strain, reorganize them into plasmids that are transformed in another strain, so an entire biosynthetic pathway can be recreated to produce a complex molecule.

Through molecular cloning, a mutant library can be generated by expressing a target plasmid in a special bacterial strain that uses an error prone polymerase when cultured at certain temperatures. The mutations can be characterized by sequencing. Bacteria transformed with mutant genes can then be tested with different drug or chemicals to see which bacterial colonies have evolved to have drug resistance.

Thanks to molecular cloning, reporter genes can be incorporated into DNA plasmids, a common reporter gene is green fluorescent protein or GFP, which emits a green fluorescence when exposed to UV light. A reporter gene can also be inserted into an alphavirus to show infection in mosquitoes and transmissibility in cells.

Youve just watched JoVEs video on molecular cloning. You should now understand how molecular cloning works and how the technique can be used in molecular biology. As always, thanks for watching!

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Large Insert Environmental Genomic Library Production

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Bacterial Transformation: The Heat Shock Method

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Restriction Enzyme Digests

Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with restriction enzymes and purified. The purified pieces are joined together though a technique called ligation. The enzyme that catalyzes the ligation reaction is known as ligase.

This video explains the major methods that are combined, in tandem, to comprise the overall molecular cloning procedure. Critical aspects of molecular cloning are discussed, such as the need for molecular cloning strategy and how to keep track of transformed bacterial colonies. Verification steps, such as checking purified plasmid for the presence of insert with restrictions digests and sequencing are also mentioned.

JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Molecular Cloning. JoVE, Cambridge, MA, doi: 10.3791/5074 (2017).

JoVE Immunology and Infection

Aaron Phillips1, Eric Mossel1, Irma Sanchez-Vargas1, Brian Foy1, Ken Olson1

1Microbiology, Immunology, and Pathology, Colorado State University

Reporter constructs can be incorporated into DNA plasmids using molecular cloning. A common reporter gene is green fluorescent protein (GFP), which emits a green fluorescence when exposed to UV light. A reporter gene was inserted into an alphavirus to show viral infection in mosquitoes and viral transmissibility in cells.

JoVE Biology

Sujata S. Jha1, Anton A. Komar1

1Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University

Here, molecular cloning is used to identify translation pause sites in mRNA in a gene of interest. The DNA template is transcribed and translated in vitro followed by the isolation and characterization of nascent polypeptides newly developed amino acid chains.

JoVE Neuroscience

Marc Bettscheider1, Arleta Kuczynska1, Osborne Almeida1, Dietmar Spengler1

1Max Planck Institute of Psychiatry

This video article shows a step-by-step protocol for examining the epigenetic modifications of genomic DNA isolated from the brains of differentially-aged mice through molecular cloning. Molecular cloning techniques are used to analyze DNA methylation of samples from the brain.

JoVE Biology

Michelle M. Denomme1,2,3, Liyue Zhang3, Mellissa R.W. Mann1,2,3

1Department of Obstretrics & Gynaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Children's Health Research Institute

The goal of this experiment is to measure DNA methylation in a single oocyte, a female germ cell, with the use of molecular cloning. Nested PCR is used to amplify the regions of DNA followed by molecular cloning to show methylation at CpG dinucleotides, sites where cytosine is next to guanine.

JoVE Biology

Marcus Taupp1, Sangwon Lee1, Alyse Hawley1, Jinshu Yang1, Steven J. Hallam1

1Department of Microbiology and Immunology, University of British Columbia - UBC

Here, researchers collected native biomass samples to isolate pieces of genomic DNA and use molecular cloning to ligate DNA fragments of appropriate size into fosmid vectors. Fosmids are cloning vectors that are based on the bacterial F (fertility)-plasmid, which can hold relatively large inserts . DNA from the transformed bacteria is packaged into virus particles to create a phage genomic DNA library.

JoVE (Journal of Visualized Experiments) is the worlds first PubMed-indexed scientific video journal. Its mission is to advance scientific research and education by increasing productivity, reproducibility, and efficiency of knowledge transfer for scientists, educators, and students worldwide through visual learning solutions.

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Molecular Cloning: Basics and Applications | Protocol

A mammoth is any species of the extinct genus Mammuthus, proboscideans commonly equipped with long, curved tusks and, in northern species, a covering of long hair. They lived from the Pliocene epoch (from around 5million years ago) into the Holocene at about 4,500 years ago[1][2] in Africa, Europe, Asia, and North America. They were members of the family Elephantidae, which also contains the two genera of modern elephants and their ancestors. Mammoths stem from an ancestral species called M. africanavus, the African mammoth. These mammoths lived in northern Africa and disappeared about 3 or 4 million years ago. Descendants of these mammoths moved north and eventually covered most of Eurasia. These were M. meridionalis, the 'southern mammoths'.[3]

The earliest known proboscideans, the clade that contains the elephants, existed about 55 million years ago around the Tethys Sea area. The closest relatives of the Proboscidea are the sirenians and the hyraxes. The family Elephantidae is known to have existed six million years ago in Africa, and includes the living elephants and the mammoths. Among many now extinct clades, the mastodon is only a distant relative of the mammoths, and part of the separate Mammutidae family, which diverged 25 million years before the mammoths evolved.[4]

The following cladogram shows the placement of the genus Mammuthus among other proboscideans, based on hyoid characteristics:[5]

Since many remains of each species of mammoth are known from several localities, it is possible to reconstruct the evolutionary history of the genus through morphological studies. Mammoth species can be identified from the number of enamel ridges on their molars; the primitive species had few ridges, and the amount increased gradually as new species evolved and replaced the former ones. At the same time, the crowns of the teeth became longer, and the skulls become higher from top to bottom and shorter from the back to the front over time to accommodate this.[6]

The first known members of the genus Mammuthus are the African species M. subplanifrons from the Pliocene and M. africanavus from the Pleistocene. The former is thought to be the ancestor of later forms. Mammoths entered Europe around 3 million years ago; the earliest known type has been named M. rumanus, which spread across Europe and China. Only its molars are known, which show it had 810 enamel ridges. A population evolved 1214 ridges and split off from and replaced the earlier type, becoming M. meridionalis. In turn, this species was replaced by the steppe mammoth, M. trogontherii, with 1820 ridges, which evolved in East Asia ca. 1 million years ago. Mammoths derived from M. trogontherii evolved molars with 26 ridges 200,000 years ago in Siberia, and became the woolly mammoth, M. primigenius.[6] The Columbian mammoth, M. columbi, evolved from a population of M. trogontherii that had entered North America. A 2011 genetic study showed that two examined specimens of the Columbian mammoth were grouped within a subclade of woolly mammoths. This suggests that the two populations interbred and produced fertile offspring. It also suggested that a North American form known as "M. jeffersonii" may be a hybrid between the two species.[7]

By the late Pleistocene, mammoths in continental Eurasia had undergone a major transformation, including a shortening and heightening of the cranium and mandible, increase in molar hypsodonty index, increase in plate number, and thinning of dental enamel. Due to this change in physical appearance, it became customary to group European mammoths separately into distinguishable clusters:

There is speculation as to what caused this variation within the three chronospecies. Variations in environment, climate change, and migration surely played roles in the evolutionary process of the mammoths. Take M. primigenius for example: Woolly mammoths lived in opened grassland biomes. The cool steppe-tundra of the Northern Hemisphere was the ideal place for mammoths to thrive because of the resources it supplied. With occasional warmings during the ice age, climate would change the landscape, and resources available to the mammoths altered accordingly.[6][8][9]

The word mammoth was first used in Europe during the early 1600s, when referring to maimanto tusks discovered in Siberia.[10] John Bell,[11] who was on the Ob River in 1722, said that mammoth tusks were well known in the area. They were called "mammon's horn" and were often found in washed-out river banks. Some local people claimed to have seen a living mammoth, but they only came out at night and always disappeared under water when detected. He bought one and presented it to Hans Sloan who pronounced it an elephant's tooth.

The folklore of some native peoples of Siberia, who would routinely find mammoth bones, and sometimes frozen mammoth bodies, in eroding river banks, had various interesting explanations for these finds. Among the Khanty people of the Irtysh River basin, a belief existed that the mammoth was some kind of a water spirit. According to other Khanty, the mammoth was a creature that lived underground, burrowing its tunnels as it went, and would die if it accidentally came to the surface.[12] The concept of the mammoth as an underground creature was known to the Chinese, who received some mammoth ivory from the Siberian natives; accordingly, the creature was known in China as yn sh , "the hidden rodent".[13]

Thomas Jefferson, who famously had a keen interest in paleontology, is partially responsible for transforming the word mammoth from a noun describing the prehistoric elephant to an adjective describing anything of surprisingly large size. The first recorded use of the word as an adjective was in a description of a large wheel of cheese (the "Cheshire Mammoth Cheese") given to Jefferson in 1802.[14]

Like their modern relatives, mammoths were quite large. The largest known species reached heights in the region of 4m (13ft) at the shoulder and weights of up to 8 tonnes (8.8 short tons), while exceptionally large males may have exceeded 12 tonnes (13 short tons). However, most species of mammoth were only about as large as a modern Asian elephant (which are about 2.5 m to 3 m high at the shoulder, and rarely exceeding 5 tonnes). Both sexes bore tusks. A first, small set appeared at about the age of six months, and these were replaced at about 18 months by the permanent set. Growth of the permanent set was at a rate of about 2.5 to 15.2cm (1 to 6in) per year.[15]

Based on studies of their close relatives, the modern elephants, mammoths probably had a gestation period of 22 months, resulting in a single calf being born. Their social structure was probably the same as that of African and Asian elephants, with females living in herds headed by a matriarch, whilst bulls lived solitary lives or formed loose groups after sexual maturity.[16]

Scientists discovered and studied the remains of a mammoth calf, and found that fat greatly influenced its form, and enabled it to store large amounts of nutrients necessary for survival in temperatures as low as 50C (58F).[17] The fat also allowed the mammoths to increase their muscle mass, allowing the mammoths to fight against enemies and live longer.[18]

Depending on the species or race of mammoth, the diet differed somewhat depending on location, although all mammoths ate similar things. For the Columbian mammoth, M. columbi, the diet was mainly grazing. American Columbian mammoths fed primarily on cacti leaves, trees, and shrubs. These assumptions were based on mammoth feces and mammoth teeth. Mammoths, like modern day elephants, have hypsodont molars. These features also allowed mammoths to live an expansive life because of the availability of grasses and trees.[19]

For the Mongochen mammoth, its diet consisted of herbs, grasses, larch, and shrubs, and possibly alder. These inferences were made through the observation of mammoth feces, which scientists observed contained non-arboreal pollen and moss spores.[20]

European mammoths had a major diet of C3 carbon fixation plants. This was determined by examining the isotopic data from the European mammoth teeth.[21]

The Yamal baby mammoth Lyuba, found in 2007 in the Yamal Peninsula in Western Siberia, suggests that baby mammoths, as do modern baby elephants, ate the dung of adult animals. The evidence to show this is that the dentition (teeth) of the baby mammoth had not yet fully developed to chew grass. Furthermore, there was an abundance of ascospores of coprophilous fungi from the pollen spectrum of the baby's mother. Coprophilous fungi are fungi that grow on animal dung and disperse spores in nearby vegetation, which the baby mammoth would then consume. Spores might have gotten into its stomach while grazing for the first few times. Coprophagy may be an adaptation, serving to populate the infant's gut with the needed microbiome for digestion.

Mammoths alive in the Arctic during the Last Glacial Maximum consumed mainly forbs, such as Artemisia; graminoids were only a minor part of their diet.[22]

The woolly mammoth (M. primigenius) was the last species of the genus. Most populations of the woolly mammoth in North America and Eurasia, as well as all the Columbian mammoths (M. columbi) in North America, died out around the time of the last glacial retreat, as part of a mass extinction of megafauna in northern Eurasia and the Americas. Until recently, the last woolly mammoths were generally assumed to have vanished from Europe and southern Siberia about 12,000 years ago, but new findings show some were still present there about 10,000 years ago. Slightly later, the woolly mammoths also disappeared from continental northern Siberia.[23] A small population survived on St. Paul Island, Alaska, up until 3750BC,[2][24][25] and the small[26] mammoths of Wrangel Island survived until 1650BC.[27][28] Recent research of sediments in Alaska indicates mammoths survived on the American mainland until 10,000 years ago.[29]

A definitive explanation for their extinction has yet to be agreed. The warming trend (Holocene) that occurred 12,000 years ago, accompanied by a glacial retreat and rising sea levels, has been suggested as a contributing factor. Forests replaced open woodlands and grasslands across the continent. The available habitat would have been reduced for some megafaunal species, such as the mammoth. However, such climate changes were nothing new; numerous very similar warming episodes had occurred previously within the ice age of the last several million years without producing comparable megafaunal extinctions, so climate alone is unlikely to have played a decisive role.[30][31] The spread of advanced human hunters through northern Eurasia and the Americas around the time of the extinctions, however, was a new development, and thus might have contributed significantly.[30][31]

Whether the general mammoth population died out for climatic reasons or due to overhunting by humans is controversial.[32] During the transition from the Late Pleistocene epoch to the Holocene epoch, there was shrinkage of the distribution of the mammoth because progressive warming at the end of the Pleistocene epoch changed the mammoth's environment. The mammoth steppe was a periglacial landscape with rich herb and grass vegetation that disappeared along with the mammoth because of environmental changes in the climate. Mammoths had moved to isolated spots in Eurasia, where they disappeared completely. Also, it is thought that Late Paleolithic and Mesolithic human hunters might have affected the size of the last mammoth populations in Europe.[citation needed] There is evidence to suggest that humans did cause the mammoth extinction, although there is no definitive proof. It was found that humans living south of a mammoth steppe learned to adapt themselves to the harsher climates north of the steppe, where mammoths resided. It was concluded that if humans could survive the harsh north climate of that particular mammoth steppe then it was possible humans could hunt (and eventually extinguish) mammoths everywhere. Another hypothesis suggests mammoths fell victim to an infectious disease. A combination of climate change and hunting by humans may be a possible explanation for their extinction. Homo erectus is known to have consumed mammoth meat as early as 1.8 million years ago,[33] though this may mean only successful scavenging, rather than actual hunting. Later humans show greater evidence for hunting mammoths; mammoth bones at a 50,000-year-old site in South Britain suggest that Neanderthals butchered the animals,[34] while various sites in Eastern Europe dating from 15,000 to 44,000 years old suggest humans (probably Homo sapiens) built dwellings using mammoth bones (the age of some of the earlier structures suggests that Neanderthals began the practice).[35] However, the American Institute of Biological Sciences also notes bones of dead elephants, left on the ground and subsequently trampled by other elephants, tend to bear marks resembling butchery marks, which have previously been misinterpreted as such by archaeologists.[citation needed]

Many hypotheses also seek to explain the regional extinction of mammoths in specific areas. Scientists have speculated that the mammoths of Saint Paul Island, an isolated enclave where mammoths survived until about 8,000 years ago, died out as the island shrank by 8090% when sea levels rose, eventually making it too small to support a viable population.[36] Similarly, genome sequences of the Wrangel Island mammoths indicate a sharp decline in genetic diversity, though the extent to which this played a role in their extinction is still unclear.[37] Another hypothesis, said to be the cause of mammoth extinction in Siberia, comes from the idea that many may have drowned. While traveling to the Northern River, many of these mammoths broke through the ice and drowned. This also explains bones remains in the Arctic Coast and islands of the New Siberian Group.[citation needed]

Dwarfing occurred with the pygmy mammoth on the outer Channel Islands of California, but at an earlier period. Those animals were very likely killed by early Paleo-Native Americans, and habitat loss caused by a rising sea level that split Santa Rosae into the outer Channel Islands.[38]

The use of preserved genetic material to create living mammoth specimens, particularly in regard to the woolly mammoth, has long been discussed theoretically but has only recently become the subject of formal effort. As of 2015, there are three major ongoing projects, one led by Akira Iritani of Japan, another by Hwang Woo-suk of South Korea, and the Long Now Foundation,[39][40] attempting to create a mammoth-elephant hybrid.[41] An estimated 150 million mammoths are buried in the Siberian tundra.[42]

In April 2015, Swedish scientists published the complete genome (complete DNA sequence) of the woolly mammoth.[43] Meanwhile, a Harvard University team is already attempting to study the animals' characteristics by inserting some mammoth genes into Asian elephant stem cells.[44] So far, the team placed mammoth genes involved in blood, fat and hair into elephant stem cells in order to study the effects of these genes in laboratory cultured cells. It is still unknown if the actual cloning of a living woolly mammoth is possible.[44]

The projects are based on finding suitable mammoth DNA in frozen bodies, sequencing its genome and, if possible, gradually combining the DNA with elephant cells.[39][40][45][46] If the cells turn viable in laboratory tests, the next challenge would be creating a viable "mammoth" hybrid embryo by inseminating an elephant egg in vitro. The percent mammoth contribution to the genome would be gradually increased on each hybrid embryo produced in vitro. If a viable hybrid embryo is obtained, it may be possible to implant it into a female Asian elephant housed in a zoo.[39] With the current knowledge and technology, it is still unlikely that the hybrid embryo would be carried through the two-year gestation.[47]

The dictionary definition of mammoth at Wiktionary

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Mammoth - Wikipedia

In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.

The term clone, invented by J. B. S. Haldane, is derived from the Ancient Greek word kln, "twig", referring to the process whereby a new plant can be created from a twig. In horticulture, the spelling clon was used until the twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o".[1][2] Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.

In botany, the term lusus was traditionally used.[3]:21, 43

Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. To amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed, and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein production, affinity tagging, single stranded RNA or DNA production and a host of other molecular biology tools.

Cloning of any DNA fragment essentially involves four steps[8]

Although these steps are invariable among cloning procedures a number of alternative routes can be selected; these are summarized as a cloning strategy.

Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector (piece of DNA). The vector (which is frequently circular) is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation, optical injection and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain colour selection markers, which provide blue/white screening (alpha-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Further investigation of the resulting colonies must be required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.

Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.

A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders).[9] In this technique a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies, each arising from a single and potentially clonal distinct cell. At an early growth stage when colonies consist of only a few cells, sterile polystyrene rings (cloning rings), which have been dipped in grease, are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

Somatic-cell nuclear transfer, known as SCNT, can also be used to create embryos for research or therapeutic purposes. The most likely purpose for this is to produce embryos for use in stem cell research. This process is also called "research cloning" or "therapeutic cloning." The goal is not to create cloned human beings (called "reproductive cloning"), but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source.[10]

Therapeutic cloning is achieved by creating embryonic stem cells in the hopes of treating diseases such as diabetes and Alzheimer's. The process begins by removing the nucleus (containing the DNA) from an egg cell and inserting a nucleus from the adult cell to be cloned.[11] In the case of someone with Alzheimer's disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus. The embryo will become genetically identical to the patient.[11] The embryo will then form a blastocyst which has the potential to form/become any cell in the body.[12]

The reason why SCNT is used for cloning is because somatic cells can be easily acquired and cultured in the lab. This process can either add or delete specific genomes of farm animals. A key point to remember is that cloning is achieved when the oocyte maintains its normal functions and instead of using sperm and egg genomes to replicate, the oocyte is inserted into the donors somatic cell nucleus.[13] The oocyte will react on the somatic cell nucleus, the same way it would on sperm cells.[13]

The process of cloning a particular farm animal using SCNT is relatively the same for all animals. The first step is to collect the somatic cells from the animal that will be cloned. The somatic cells could be used immediately or stored in the laboratory for later use.[13] The hardest part of SCNT is removing maternal DNA from an oocyte at metaphase II. Once this has been done, the somatic nucleus can be inserted into an egg cytoplasm.[13] This creates a one-cell embryo. The grouped somatic cell and egg cytoplasm are then introduced to an electrical current.[13] This energy will hopefully allow the cloned embryo to begin development. The successfully developed embryos are then placed in surrogate recipients, such as a cow or sheep in the case of farm animals.[13]

SCNT is seen as a good method for producing agriculture animals for food consumption. It successfully cloned sheep, cattle, goats, and pigs. Another benefit is SCNT is seen as a solution to clone endangered species that are on the verge of going extinct.[13] However, stresses placed on both the egg cell and the introduced nucleus can be enormous, which led to a high loss in resulting cells in early research. For example, the cloned sheep Dolly was born after 277 eggs were used for SCNT, which created 29 viable embryos. Only three of these embryos survived until birth, and only one survived to adulthood.[14] As the procedure could not be automated, and had to be performed manually under a microscope, SCNT was very resource intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg was also far from being well-understood. However, by 2014 researchers were reporting cloning success rates of seven to eight out of ten[15] and in 2016, a Korean Company Sooam Biotech was reported to be producing 500 cloned embryos per day.[16]

In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus.

Organism cloning (also called reproductive cloning) refers to the procedure of creating a new multicellular organism, genetically identical to another. In essence this form of cloning is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction is a naturally occurring phenomenon in many species, including most plants (see vegetative reproduction) and some insects. Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However, cloning, or asexual propagation,[17] has been common practice in the horticultural world for hundreds of years.

The term clone is used in horticulture to refer to descendants of a single plant which were produced by vegetative reproduction or apomixis. Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction.[18] As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana.[19]Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operation.

Many trees, shrubs, vines, ferns and other herbaceous perennials form clonal colonies naturally. Parts of an individual plant may become detached by fragmentation and grow on to become separate clonal individuals. A common example is in the vegetative reproduction of moss and liverwort gametophyte clones by means of gemmae. Some vascular plants e.g. dandelion and certain viviparous grasses also form seeds asexually, termed apomixis, resulting in clonal populations of genetically identical individuals.

Clonal derivation exists in nature in some animal species and is referred to as parthenogenesis (reproduction of an organism by itself without a mate). This is an asexual form of reproduction that is only found in females of some insects, crustaceans, nematodes,[20] fish (for example the hammerhead shark[21]), the Komodo dragon[21] and lizards. The growth and development occurs without fertilization by a male. In plants, parthenogenesis means the development of an embryo from an unfertilized egg cell, and is a component process of apomixis. In species that use the XY sex-determination system, the offspring will always be female. An example is the little fire ant (Wasmannia auropunctata), which is native to Central and South America but has spread throughout many tropical environments.

Artificial cloning of organisms may also be called reproductive cloning.

Hans Spemann, a German embryologist was awarded a Nobel Prize in Physiology or Medicine in 1935 for his discovery of the effect now known as embryonic induction, exercised by various parts of the embryo, that directs the development of groups of cells into particular tissues and organs. In 1928 he and his student, Hilde Mangold, were the first to perform somatic-cell nuclear transfer using amphibian embryos one of the first moves towards cloning.[22]

Reproductive cloning generally uses "somatic cell nuclear transfer" (SCNT) to create animals that are genetically identical. This process entails the transfer of a nucleus from a donor adult cell (somatic cell) to an egg from which the nucleus has been removed, or to a cell from a blastocyst from which the nucleus has been removed.[23] If the egg begins to divide normally it is transferred into the uterus of the surrogate mother. Such clones are not strictly identical since the somatic cells may contain mutations in their nuclear DNA. Additionally, the mitochondria in the cytoplasm also contains DNA and during SCNT this mitochondrial DNA is wholly from the cytoplasmic donor's egg, thus the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to death.

Artificial embryo splitting or embryo twinning, a technique that creates monozygotic twins from a single embryo, is not considered in the same fashion as other methods of cloning. During that procedure, an donor embryo is split in two distinct embryos, that can then be transferred via embryo transfer. It is optimally performed at the 6- to 8-cell stage, where it can be used as an expansion of IVF to increase the number of available embryos.[24] If both embryos are successful, it gives rise to monozygotic (identical) twins.

Dolly, a Finn-Dorset ewe, was the first mammal to have been successfully cloned from an adult somatic cell. Dolly was formed by taking a cell from the udder of her 6-year old biological mother.[25] Dolly's embryo was created by taking the cell and inserting it into a sheep ovum. It took 434 attempts before an embryo was successful.[26] The embryo was then placed inside a female sheep that went through a normal pregnancy.[27] She was cloned at the Roslin Institute in Scotland by British scientists Sir Ian Wilmut and Keith Campbell and lived there from her birth in 1996 until her death in 2003 when she was six. She was born on 5 July 1996 but not announced to the world until 22 February 1997.[28] Her stuffed remains were placed at Edinburgh's Royal Museum, part of the National Museums of Scotland.[29]

Dolly was publicly significant because the effort showed that genetic material from a specific adult cell, programmed to express only a distinct subset of its genes, can be reprogrammed to grow an entirely new organism. Before this demonstration, it had been shown by John Gurdon that nuclei from differentiated cells could give rise to an entire organism after transplantation into an enucleated egg.[30] However, this concept was not yet demonstrated in a mammalian system.

The first mammalian cloning (resulting in Dolly the sheep) had a success rate of 29 embryos per 277 fertilized eggs, which produced three lambs at birth, one of which lived. In a bovine experiment involving 70 cloned calves, one-third of the calves died young. The first successfully cloned horse, Prometea, took 814 attempts. Notably, although the first[clarification needed] clones were frogs, no adult cloned frog has yet been produced from a somatic adult nucleus donor cell.

There were early claims that Dolly the sheep had pathologies resembling accelerated aging. Scientists speculated that Dolly's death in 2003 was related to the shortening of telomeres, DNA-protein complexes that protect the end of linear chromosomes. However, other researchers, including Ian Wilmut who led the team that successfully cloned Dolly, argue that Dolly's early death due to respiratory infection was unrelated to deficiencies with the cloning process. This idea that the nuclei have not irreversibly aged was shown in 2013 to be true for mice.[31]

Dolly was named after performer Dolly Parton because the cells cloned to make her were from a mammary gland cell, and Parton is known for her ample cleavage.[32]

The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Notable experiments include:

Human cloning is the creation of a genetically identical copy of a human. The term is generally used to refer to artificial human cloning, which is the reproduction of human cells and tissues. It does not refer to the natural conception and delivery of identical twins. The possibility of human cloning has raised controversies. These ethical concerns have prompted several nations to pass legislature regarding human cloning and its legality.

Two commonly discussed types of theoretical human cloning are therapeutic cloning and reproductive cloning. Therapeutic cloning would involve cloning cells from a human for use in medicine and transplants, and is an active area of research, but is not in medical practice anywhere in the world, as of 2014. Two common methods of therapeutic cloning that are being researched are somatic-cell nuclear transfer and, more recently, pluripotent stem cell induction. Reproductive cloning would involve making an entire cloned human, instead of just specific cells or tissues.[57]

There are a variety of ethical positions regarding the possibilities of cloning, especially human cloning. While many of these views are religious in origin, the questions raised by cloning are faced by secular perspectives as well. Perspectives on human cloning are theoretical, as human therapeutic and reproductive cloning are not commercially used; animals are currently cloned in laboratories and in livestock production.

Advocates support development of therapeutic cloning in order to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants,[58] to avoid the need for immunosuppressive drugs,[57] and to stave off the effects of aging.[59] Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology.[60]

Opponents of cloning have concerns that technology is not yet developed enough to be safe[61] and that it could be prone to abuse (leading to the generation of humans from whom organs and tissues would be harvested),[62][63] as well as concerns about how cloned individuals could integrate with families and with society at large.[64][65]

Religious groups are divided, with some opposing the technology as usurping "God's place" and, to the extent embryos are used, destroying a human life; others support therapeutic cloning's potential life-saving benefits.[66][67]

Cloning of animals is opposed by animal-groups due to the number of cloned animals that suffer from malformations before they die,[68][69] and while food from cloned animals has been approved by the US FDA,[70][71] its use is opposed by groups concerned about food safety.[72][73][74]

Cloning, or more precisely, the reconstruction of functional DNA from extinct species has, for decades, been a dream. Possible implications of this were dramatized in the 1984 novel Carnosaur and the 1990 novel Jurassic Park.[75][76] The best current cloning techniques have an average success rate of 9.4 percent[77] (and as high as 25 percent[31]) when working with familiar species such as mice,[note 1] while cloning wild animals is usually less than 1 percent successful.[80] Several tissue banks have come into existence, including the "Frozen Zoo" at the San Diego Zoo, to store frozen tissue from the world's rarest and most endangered species.[75][81][82]

In 2001, a cow named Bessie gave birth to a cloned Asian gaur, an endangered species, but the calf died after two days. In 2003, a banteng was successfully cloned, followed by three African wildcats from a thawed frozen embryo. These successes provided hope that similar techniques (using surrogate mothers of another species) might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo (Pyrenean ibex) were frozen in liquid nitrogen immediately after it died in 2000. Researchers are also considering cloning endangered species such as the giant panda and cheetah.

In 2002, geneticists at the Australian Museum announced that they had replicated DNA of the thylacine (Tasmanian tiger), at the time extinct for about 65 years, using polymerase chain reaction.[83] However, on 15 February 2005 the museum announced that it was stopping the project after tests showed the specimens' DNA had been too badly degraded by the (ethanol) preservative. On 15 May 2005 it was announced that the thylacine project would be revived, with new participation from researchers in New South Wales and Victoria.

In January 2009, for the first time, an extinct animal, the Pyrenean ibex mentioned above was cloned, at the Centre of Food Technology and Research of Aragon, using the preserved frozen cell nucleus of the skin samples from 2001 and domestic goat egg-cells. The ibex died shortly after birth due to physical defects in its lungs.[84]

One of the most anticipated targets for cloning was once the woolly mammoth, but attempts to extract DNA from frozen mammoths have been unsuccessful, though a joint Russo-Japanese team is currently working toward this goal. In January 2011, it was reported by Yomiuri Shimbun that a team of scientists headed by Akira Iritani of Kyoto University had built upon research by Dr. Wakayama, saying that they will extract DNA from a mammoth carcass that had been preserved in a Russian laboratory and insert it into the egg cells of an African elephant in hopes of producing a mammoth embryo. The researchers said they hoped to produce a baby mammoth within six years.[85][86] It was noted, however that the result, if possible, would be an elephant-mammoth hybrid rather than a true mammoth.[87] Another problem is the survival of the reconstructed mammoth: ruminants rely on a symbiosis with specific microbiota in their stomachs for digestion.[87]

Scientists at the University of Newcastle and University of New South Wales announced in March 2013 that the very recently extinct gastric-brooding frog would be the subject of a cloning attempt to resurrect the species.[88]

Many such "de-extinction" projects are described in the Long Now Foundation's Revive and Restore Project.[89]

After an eight-year project involving the use of a pioneering cloning technique, Japanese researchers created 25 generations of healthy cloned mice with normal lifespans, demonstrating that clones are not intrinsically shorter-lived than naturally born animals.[31][90]

In a detailed study released in 2016 and less detailed studies by others suggest that once cloned animals get past the first month or two of life they are generally healthy. However, early pregnancy loss and neonatal losses are still greater with cloning than natural conception or assisted reproduction (IVF). Current research endeavors are attempting to overcome this problem.[32]

In an article in the 8 November 1993 article of Time, cloning was portrayed in a negative way, modifying Michelangelo's Creation of Adam to depict Adam with five identical hands. Newsweek's 10 March 1997 issue also critiqued the ethics of human cloning, and included a graphic depicting identical babies in beakers.

Cloning is a recurring theme in a wide variety of contemporary science fiction, ranging from action films such as Jurassic Park (1993), The 6th Day (2000), Resident Evil (2002), Star Wars (2002) and The Island (2005), to comedies such as Woody Allen's 1973 film Sleeper.[91]

Science fiction has used cloning, most commonly and specifically human cloning, due to the fact that it brings up controversial questions of identity.[92][93]A Number is a 2002 play by English playwright Caryl Churchill which addresses the subject of human cloning and identity, especially nature and nurture. The story, set in the near future, is structured around the conflict between a father (Salter) and his sons (Bernard 1, Bernard 2, and Michael Black) two of whom are clones of the first one. A Number was adapted by Caryl Churchill for television, in a co-production between the BBC and HBO Films.[94]

A recurring sub-theme of cloning fiction is the use of clones as a supply of organs for transplantation. The 2005 Kazuo Ishiguro novel Never Let Me Go and the 2010 film adaption[95] are set in an alternate history in which cloned humans are created for the sole purpose of providing organ donations to naturally born humans, despite the fact that they are fully sentient and self-aware. The 2005 film The Island[96] revolves around a similar plot, with the exception that the clones are unaware of the reason for their existence.

The use of human cloning for military purposes has also been explored in several works. Star Wars portrays human cloning in Clone Wars.[97]

The exploitation of human clones for dangerous and undesirable work was examined in the 2009 British science fiction film Moon.[98] In the futuristic novel Cloud Atlas and subsequent film, one of the story lines focuses on a genetically-engineered fabricant clone named Sonmi~451 who is one of millions raised in an artificial "wombtank," destined to serve from birth. She is one of thousands of clones created for manual and emotional labor; Sonmi herself works as a server in a restaurant. She later discovers that the sole source of food for clones, called 'Soap', is manufactured from the clones themselves.[99]

Cloning has been used in fiction as a way of recreating historical figures. In the 1976 Ira Levin novel The Boys from Brazil and its 1978 film adaptation, Josef Mengele uses cloning to create copies of Adolf Hitler.[100]

In 2012, a Japanese television show named "Bunshin" was created. The story's main character, Mariko, is a woman studying child welfare in Hokkaido. She grew up always doubtful about the love from her mother, who looked nothing like her and who died nine years before. One day, she finds some of her mother's belongings at a relative's house, and heads to Tokyo to seek out the truth behind her birth. She later discovered that she was a clone.[101]

In the 2013 television show Orphan Black, cloning is used as a scientific study on the behavioral adaptation of the clones.[102] In a similar vein, the book The Double by Nobel Prize winner Jos Saramago explores the emotional experience of a man who discovers that he is a clone.[103]

Originally posted here:

Cloning - Wikipedia

Cloning/Embryonic Stem Cells

The term cloning is used by scientists to describe many different processes that involve making duplicates of biological material. In most cases, isolated genes or cells are duplicated for scientific study, and no new animal results. The experiment that led to the cloning of Dolly the sheep in 1997 was different: It used a cloning technique called somatic cell nuclear transfer and resulted in an animal that was a genetic twin -- although delayed in time -- of an adult sheep. This technique can also be used to produce an embryo from which cells called embryonic stem (ES) cells could be extracted to use in research into potential therapies for a wide variety of diseases.

Thus, in the past five years, much of the scientific and ethical debate about somatic cell nuclear transfer has focused on its two potential applications: 1) for reproductive purposes, i.e., to produce a child, or 2) for producing a source of ES cells for research.

The technique of transferring a nucleus from a somatic cell into an egg that produced Dolly was an extension of experiments that had been ongoing for over 40 years. In the simplest terms, the technique used to produce Dolly the sheep - somatic cell nuclear transplantation cloning - involves removing the nucleus of an egg and replacing it with the diploid nucleus of a somatic cell. Unlike sexual reproduction, during which a new organism is formed when the genetic material of the egg and sperm fuse, in nuclear transplantation cloning there is a single genetic "parent." This technique also differs from previous cloning techniques because it does not involve an existing embryo. Dolly is different because she is not genetically unique; when born she was genetically identical to an existing six-year-old ewe. Although the birth of Dolly was lauded as a success, in fact, the procedure has not been perfected and it is not yet clear whether Dolly will remain healthy or whether she is already experiencing subtle problems that might lead to serious diseases. Thus, the prospect of applying this technique in humans is troubling for scientific and safety reasons in addition to a variety of ethical reasons related to our ideas about the natural ordering of family and successive generations.

Several important concerns remain about the science and safety of nuclear transfer cloning using adult cells as the source of nuclei. To date, five mammalian species -- sheep, cattle, pigs, goats, and mice -- have been used extensively in reproductive cloning studies. Data from these experiments illustrate the problems involved. Typically, very few cloning attempts are successful. Many cloned animals die in utero, even at late stages or soon after birth, and those that survive frequently exhibit severe birth defects. In addition, female animals carrying cloned fetuses may face serious risks, including death from cloning-related complications.

An additional concern focuses on whether cellular aging will affect the ability of somatic cell nuclei to program normal development. As somatic cells divide they progressively age, and there is normally a defined number of cell divisions that can occur before senescence. Thus, the health effects for the resulting liveborn, having been created with an "aged" nucleus, are unknown. Recently it was reported that Dolly has arthritis, although it is not yet clear whether the five-and-a-half-year-old sheep is suffering from the condition as a result of the cloning process. And, scientists in Tokyo have shown that cloned mice die significantly earlier than those that are naturally conceived, raising an additional concern that the mutations that accumulate in somatic cells might affect nuclear transfer efficiency and lead to cancer and other diseases in offspring. Researchers working with clones of a Holstein cow say genetic programming errors may explain why so many cloned animals die, either as fetuses or newborns.

The announcement of Dolly sparked widespread speculation about a human child being created using somatic cell nuclear transfer. Much of the perceived fear that greeted this announcement centered on the misperception that a child or many children could be produced who would be identical to an already existing person. This fear is based on the idea of "genetic determinism" -- that genes alone determine all aspects of an individual -- and reflects the belief that a person's genes bear a simple relationship to the physical and psychological traits that compose that individual. Although genes play an essential role in the formation of physical and behavioral characteristics, each individual is, in fact, the result of a complex interaction between his or her genes and the environment within which he or she develops. Nonetheless, many of the concerns about cloning have focused on issues related to "playing God," interfering with the natural order of life, and somehow robbing a future individual of the right to a unique identity.

Several groups have concluded that reproductive cloning of human beings creates ethical and scientific risks that society should not tolerate. In 1997, the National Bioethics Advisory Commission recommended that it was morally unacceptable to attempt to create a child using somatic cell nuclear transfer cloning and suggested that a moratorium be imposed until safety of this technique could be assessed. The commission also cautioned against preempting the use of cloning technology for purposes unrelated to producing a liveborn child.

Similarly, in 2001 the National Academy of Sciences issued a report stating that the United States should ban human reproductive cloning aimed at creating a child because experience with reproductive cloning in animals suggests that the process would be dangerous for the woman, the fetus, and the newborn, and would likely fail. The report recommended that the proposed ban on human cloning should be reviewed within five years, but that it should be reconsidered "only if a new scientific review indicates that the procedures are likely to be safe and effective, and if a broad national dialogue on societal, religious and ethical issues suggests that reconsideration is warranted." The panel concluded that the scientific and medical considerations that justify a ban on human reproductive cloning at this time do not apply to nuclear transplantation to produce stem cells. Several other scientific and medical groups also have stated their opposition to the use of cloning for the purpose of producing a child.

The cloning debate was reopened with a new twist late in 1998, when two scientific reports were published regarding the successful isolation of human stem cells. Stem cells are unique and essential cells found in animals that are capable of continually reproducing themselves and renewing tissue throughout an individual organism's life. ES cells are the most versatile of all stem cells because they are less differentiated, or committed, to a particular function than adult stem cells. These cells have offered hope of new cures to debilitating and even fatal illness. Recent studies in mice and other animals have shown that ES cells can reduce symptoms of Parkinson's disease in mouse models, and work in other animal models and disease areas seems promising.

In the 1998 reports, ES cells were derived from in vitro embryos six to seven days old destined to be discarded by couples undergoing infertility treatments, and embryonic germ (EG) cells were obtained from cadaveric fetal tissue following elective abortion. A third report, appearing in the New York Times, claimed that a Massachusetts biotechnology company had fused a human cell with an enucleated cow egg, creating a hybrid clone that failed to progress beyond an early stage of development. This announcement served as a reminder that ES cells also could be derived from embryos created through somatic cell nuclear transfer, or cloning. In fact, several scientists believed that deriving ES cells in this manner is the most promising approach to developing treatments because the condition of in vitro fertilization (IVF) embryos stored over time is questionable and this type of cloning could overcome graft-host responses if resulting therapies were developed from the recipient's own DNA.

For those who believe that the embryo has the moral status of a person from the moment of conception, research or any other activity that would destroy it is wrong. For those who believe the human embryo deserves some measure of respect, but disagree that the respect due should equal that given to a fully formed human, it could be considered immoral not to use embryos that would otherwise be destroyed to develop potential cures for disease affecting millions of people. An additional concern related to public policy is whether federal funds should be used for research that some Americans find unethical.

Since 1996, Congress has prohibited researchers from using federal funds for human embryo research. In 1999, DHHS announced that it intended to fund research on human ES cells derived from embryos remaining after infertility treatments. This decision was based on an interpretation "that human embryonic stem cells are not a human embryo within the statutory definition" because "the cells do not have the capacity to develop into a human being even if transferred to the uterus, thus their destruction in the course of research would not constitute the destruction of an embryo." DHHS did not intend to fund research using stem cells derived from embryos created through cloning, although such efforts would be legal in the private sector.

In July 2001, the House of Representatives voted 265 to 162 to make any human cloning a criminal offense, including cloning to create an embryo for derivation of stem cells rather than to produce a child. In August 2002, President Bush, contending with a DHHS decision made during the Clinton administration, stated in a prime-time television address that federal support would be provided for research using a limited number of stem cell colonies already in existence (derived from leftover IVF embryos). Current bills before Congress would ban all forms of cloning outright, prohibit cloning for reproductive purposes, and impose a moratorium on cloning to derive stem cells for research, or prohibit cloning for reproductive purposes while allowing cloning for therapeutic purposes to go forward. As of late June, the Senate has taken no action. President Bush's Bioethics Council is expected to recommend the prohibition of reproductive cloning and a moratorium on therapeutic cloning later this summer.

Prepared by Kathi E. Hanna, M.S., Ph.D., Science and Health Policy Consultant

Last Reviewed: April 2006

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Cloning/Embryonic Stem Cells - National Human Genome Research ...

Cloning is the process of creating a copy of a biological entity. In genetics, it refers to the process of making an identical copy of the DNA of an organism. Are you interested in understanding the pros and cons of cloning?

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When Dolly, the first cloned sheep came in the news, cloning interested the masses. Not only researchers but even common people became interested in knowing about how cloning is done and what pros and cons it has. Everyone became more curious about how cloning could benefit the common man. Most of us want to know the pros and cons of cloning, its advantages and its potential risks to mankind. Let us understand them.

Cloning finds applications in genetic fingerprinting, amplification of DNA and alteration of the genetic makeup of organisms. It can be used to bring about desired changes in the genetic makeup of individuals thereby introducing positive traits in them, as also for the elimination of negative traits. Cloning can also be applied to plants to remove or alter defective genes, thereby making them resistant to diseases. Cloning may find applications in the development of human organs, thus making human life safer. Here we look at some of the potential advantages of cloning.

Organ Replacement

If vital organs of the human body can be cloned, they can serve as backups. Cloning body parts can serve as a lifesaver. When a body organ such as a kidney or heart fails to function, it may be possible to replace it with the cloned body organ.

Substitute for Natural Reproduction

Cloning in human beings can prove to be a solution to infertility. It can serve as an option for producing children. With cloning, it would be possible to produce certain desired traits in human beings. We might be able to produce children with certain qualities. Wouldn't that be close to creating a man-made being?!

Help in Genetic Research

Cloning technologies can prove helpful to researchers in genetics. They might be able to understand the composition of genes and the effects of genetic constituents on human traits, in a better manner. They will be able to alter genetic constituents in cloned human beings, thus simplifying their analysis of genes. Cloning may also help us combat a wide range of genetic diseases.

Obtain Specific Traits in Organisms

Cloning can make it possible for us to obtain customized organisms and harness them for the benefit of society. It can serve as the best means to replicate animals that can be used for research purposes. It can enable the genetic alteration of plants and animals. If positive changes can be brought about in living beings with the help of cloning, it will indeed be a boon to mankind.

Like every coin has two sides, cloning has its flip side too. Though cloning may work wonders in genetics, it has some potential disadvantages. Cloning, as you know, is copying or replicating biological traits in organisms. Thus it might reduce the diversity in nature. Imagine multiple living entities like one another! Another con of cloning is that it is not clear whether we will be able to bring all the potential uses of cloning into reality. Plus, there's a big question of whether the common man will afford harnessing cloning technologies to his benefit. Here we look at the potential disadvantages of cloning.

Detrimental to Genetic Diversity

Cloning creates identical genes. It is a process of replicating a genetic constitution, thus hampering the diversity in genes. In lessening genetic diversity, we weaken our ability of adaptation. Cloning is also detrimental to the beauty that lies in diversity.

Invitation to Malpractices

While cloning allows man to tamper with genes in human beings, it also makes deliberate reproduction of undesirable traits, a possibility. Cloning of body organs may invite malpractices in society.

Will it Reach the Common Man?

In cloning human organs and using them for transplant, or in cloning human beings themselves, technical and economic barriers will have to be considered. Will cloned organs be cost-effective? Will cloning techniques really reach the common man?

Man, a Man-made Being?

Moreover, cloning will put human and animal rights at stake. Will cloning fit into our ethical and moral principles? It will make man just another man-made being. Won't it devalue mankind? Won't it demean the value of human life?

Cloning is equal to emulating God. Is that easy? Is it risk-free? Many are afraid it is not.

Manali Oak

Last Updated: August 8, 2016

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Pros and Cons of Cloning - Buzzle

Dolly (5 July 1996 14 February 2003) was a female domestic sheep, and the first mammal cloned from an adult somatic cell, using the process of nuclear transfer.[2][3] She was cloned by Sir Ian Wilmut, Keith Campbell and colleagues at the Roslin Institute, part of the University of Edinburgh, Scotland, and the biotechnology company PPL Therapeutics, based near Edinburgh. The funding for Dolly's cloning was provided by PPL Therapeutics and the UK's Ministry of Agriculture.[4] She was born on 5 July 1996 and died from a progressive lung disease 5 months before her seventh birthday.[5] She has been called "the world's most famous sheep" by sources including BBC News and Scientific American.[6][7]

The cell used as the donor for the cloning of Dolly was taken from a mammary gland, and the production of a healthy clone therefore proved that a cell taken from a specific part of the body could recreate a whole individual. On Dolly's name, Wilmut stated "Dolly is derived from a mammary gland cell and we couldn't think of a more impressive pair of glands than Dolly Parton's".[1]

Dolly was born on 5 July 1996 and had three mothers (one provided the egg, another the DNA and a third carried the cloned embryo to term).[8] She was created using the technique of somatic cell nuclear transfer, where the cell nucleus from an adult cell is transferred into an unfertilized oocyte (developing egg cell) that has had its cell nucleus removed. The hybrid cell is then stimulated to divide by an electric shock, and when it develops into a blastocyst it is implanted in a surrogate mother.[9] Dolly was the first clone produced from a cell taken from an adult mammal. The production of Dolly showed that genes in the nucleus of such a mature differentiated somatic cell are still capable of reverting to an embryonic totipotent state, creating a cell that can then go on to develop into any part of an animal.[10] Dolly's existence was announced to the public on 22 February 1997.[1] It gained much attention in the media. A commercial with Scottish scientists playing with sheep was aired on TV, and a special report in TIME Magazine featured Dolly the sheep.[4]Science featured Dolly as the breakthrough of the year. Even though Dolly was not the first animal cloned, she received media attention because she was the first cloned from an adult cell.[11]

Dolly lived her entire life at the Roslin Institute in Edinburgh. There she was bred with a Welsh Mountain ram and produced six lambs in total. Her first lamb, named Bonnie, was born in April 1998.[5] The next year Dolly produced twin lambs Sally and Rosie, and she gave birth to triplets Lucy, Darcy and Cotton in the year after that.[12] In late 2001, at the age of four, Dolly developed arthritis and began to walk stiffly. This was treated with anti-inflammatory drugs.[13]

On 14 February 2003, Dolly was euthanised because she had a progressive lung disease and severe arthritis.[14] A Finn Dorset such as Dolly has a life expectancy of around 11 to 12 years, but Dolly lived 6.5 years. A post-mortem examination showed she had a form of lung cancer called Jaagsiekte,[15] which is a fairly common disease of sheep and is caused by the retrovirus JSRV.[16] Roslin scientists stated that they did not think there was a connection with Dolly being a clone, and that other sheep in the same flock had died of the same disease.[14] Such lung diseases are a particular danger for sheep kept indoors, and Dolly had to sleep inside for security reasons.

Some in the press speculated that a contributing factor to Dolly's death was that she could have been born with a genetic age of six years, the same age as the sheep from which she was cloned.[17] One basis for this idea was the finding that Dolly's telomeres were short, which is typically a result of the aging process.[18][19] The Roslin Institute stated that intensive health screening did not reveal any abnormalities in Dolly that could have come from advanced aging.[17]

In 2016 scientists reported no defects in thirteen cloned sheep, including four from the same cell line as Dolly. The first study to review the long-term health outcomes of cloning, the authors found no evidence of late-onset, non-communicable diseases other than some minor examples of oseteoarthritis and concluded "We could find no evidence, therefore, of a detrimental long-term effect of cloning by SCNT on the health of aged offspring among our cohort."[20][21]

After cloning was successfully demonstrated through the production of Dolly, many other large mammals were cloned, including pigs,[22][23]deer,[24]horses[25] and bulls.[26] The attempt to clone argali (mountain sheep) did not produce viable embryos. The attempt to clone a banteng bull was more successful, as were the attempts to clone mouflon (a form of wild sheep), both resulting in viable offspring.[27] The reprogramming process cells need to go through during cloning is not perfect and embryos produced by nuclear transfer often show abnormal development.[28][29] Making cloned mammals was highly inefficient in 1996 Dolly was the only lamb that survived to adulthood from 277 attempts. However, by 2014 Chinese scientists were reported to have 7080% success rates cloning pigs[23] and in 2016, a Korean company, Sooam Biotech was producing 500 cloned embryos a day.[30] Wilmut, who led the team that created Dolly, announced in 2007 that the nuclear transfer technique may never be sufficiently efficient for use in humans.[31]

Cloning may have uses in preserving endangered species and may become a viable tool for reviving extinct species.[32] In January 2009, scientists from the Centre of Food Technology and Research of Aragon, in northern Spain announced the cloning of the Pyrenean ibex, a form of wild mountain goat, which was officially declared extinct in 2000. Although the newborn ibex died shortly after birth due to physical defects in its lungs, it is the first time an extinct animal has been cloned, and may open doors for saving endangered and newly extinct species by resurrecting them from frozen tissue.[33][34]

In July, 2016, four identical clones of the Dolly sheep (Daisy, Debbie, Dianna and Denise) were alive and healthy at nine years old.[35][36]

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Dolly (sheep) - Wikipedia, the free encyclopedia

The essence of cell chemistry is to isolate a particular cellular component and then analyze its chemical structure and activity. In the case of DNA, this is feasible for relatively short molecules such as the genomes of small viruses. But genomes of even the simplest cells are much too large to directly analyze in detail at the molecular level. The problem is compounded for complex organisms. The human genome, for example, contains about 6 109base pairs (bp) in the 23 pairs of chromosomes. Cleavage of human DNA with restriction enzymes that produce about one cut for every 3000 base pairs yields some 2 million fragments, far too many to separate from each other directly. This obstacle to obtaining pure DNA samples from large genomes has been overcome by recombinant DNA technology. With these methods virtually any gene can be purified, its sequence determined, and the functional regions of the sequence explored by altering it in planned ways and reintroducing the DNA into cells and into whole organisms.

The essence of recombinant DNA technology is the prep-aration of large numbers of identical DNA molecules. A DNA fragment of interest is linked through standard 35 phosphodiester bonds to a vector DNA molecule, which can replicate when introduced into a host cell. When a single recombinant DNA molecule, composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the inserted DNA is reproduced along with the vector, producing large numbers of recombinant DNA molecules that include the fragment of DNA originally linked to the vector. Two types of vectors are most commonly used: E. coli plasmid vectors and bacteriophage vectors. Plasmid vectors replicate along with their host cells, while vectors replicate as lytic viruses, killing the host cell and packaging the DNA into virions (Chapter 6). In this section, the general procedure for cloning DNA fragments in E. coli plasmids is described.

Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cells chromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria, yeast, and some higher eukaryotic cells, exist in a parasitic or symbiotic relationship with their host cell. Plasmids range in size from a few thousand base pairs to more than 100 kilobases (kb). Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell.

Many naturally occurring plasmids contain genes that provide some benefit to the host cell, fulfilling the plasmids portion of the symbiotic relationship. For example, some bacterial plasmids encode enzymes that inactivate antibiotics. Such drug-resistance plasmids have become a major problem in the treatment of a number of common bacterial pathogens. As antibiotic use became widespread, plasmids containing several drug-resistance genes evolved, making their host cells resistant to a variety of different antibiotics simultaneously. Many of these plasmids also contain transfer genes encoding proteins that can form a macromolecular tube, or pilus, through which a copy of the plasmid can be transferred to other host cells of the same or related bacterial species. Such transfer can result in the rapid spread of drug-resistance plasmids, expanding the number of antibiotic-resistant bacteria in an environment such as a hospital. Coping with the spread of drug-resistance plasmids is an important challenge for modern medicine.

The plasmids most commonly used in recombinant DNA technology replicate in E. coli.Generally, these plasmids have been engineered to optimize their use as vectors in DNA cloning. For instance, to simplify working with plasmids, their length is reduced; many plasmid vectors are only 3kb in length, which is much shorter than in naturally occurring E. coli plasmids. (The circumference of plasmids usually is referred to as their length, even though plasmids are almost always circular DNA molecules.) Most plasmid vectors contain little more than the essential nucleotide sequences required for their use in DNA cloning: a replication origin, a drug-resistance gene, and a region in which exogenous DNA fragments can be inserted ().

Diagram of a simple cloning vector derived from a plasmid, a circular, double-stranded DNA molecule that can replicate within an E. coli cell. Plasmid vectors are 1.23 kb in length and contain a replication origin (more...)

The replication origin (ORI) is a specific DNA sequence of 50100 base pairs that must be present in a plasmid for it to replicate. Host-cell enzymes bind to ORI, initiating replication of the circular plasmid. Once DNA replication is initiated at ORI, it continues around the circular plasmid regardless of its nucleotide sequence (). Thus any DNA sequence inserted into such a plasmid is replicated along with the rest of the plasmid DNA; this property is the basis of molecular DNA cloning.

Plasmid DNA replication. The parental strands are shown in blue, and newly synthesized daughter strands are shown in red. The short segments represent the AT and GC base pairs connecting the complementary strands. Once DNA replication (more...)

In 1944, O. T. Avery, C. M. Macleod, and M. McCarty first demonstrated gene transfer with isolated DNA obtained from Streptococcus pneumoniae. This process involved the genetic alteration of a bacterial cell by the uptake of DNA isolated from a genetically different bacterium and its recombination with the host-cell genome. Their experiments provided the first evidence that DNA is the genetic material. Later studies showed that such genetic alteration of a recipient cell can result from the uptake of exogenous extrachromosomal DNA (e.g., plasmids) that does not integrate into the host-cell chromosome. The term transformation is used to denote the genetic alteration of a cell caused by the uptake and expression of foreign DNA regardless of the mechanism involved. (Note that transformation has a second meaning defined in Chapter 6, namely, the process by which normal cells with a finite life span in culture are converted into continuously growing cells similar to cancer cells.)

The phenomenon of transformation permits plasmid vectors to be introduced into and expressed by E. coli cells. In order to be useful in DNA cloning, however, a plasmid vector must contain a selectable gene, most commonly a drug-resistance gene encoding an enzyme that inactivates a specific antibiotic. As weve seen, the ampicillin-resistance gene (ampr) encodes -lactamase, which inactivates the antibiotic ampicillin. After plasmid vectors are incubated with E. coli, those cells that take up the plasmid can be easily selected from the larger number of cells that do not by growing them in an ampicillin-containing medium. The ability to select transformed cells is critical to DNA cloning by plasmid vector technology because the transformation of E. coli with isolated plasmid DNA is inefficient.

Normal E. coli cells cannot take up plasmid DNA from the medium. Exposure of cells to high concentrations of certain divalent cations, however, makes a small fraction of cells permeable to foreign DNA by a mechanism that is not understood. In a typical procedure, E. coli cells are treated with CaCl2 and mixed with plasmid vectors; commonly, only 1 cell in about 10,000 or more cells becomes competent to take up the foreign DNA. Each competent cell incorporates a single plasmid DNA molecule, which carries an antibiotic-resistance gene. When the treated cells are plated on a petri dish of nutrient agar containing the antibiotic, only the rare transformed cells containing the antibiotic-resistance gene on the plasmid vector will survive. All the plasmids in such a colony of selected transformed cells are descended from the single plasmid taken up by the cell that established the colony.

A DNA fragment of a few base pairs up to 20 kb can be inserted into a plasmid vector. When such a recombinant plasmid transforms an E. coli cell, all the antibiotic-resistant progeny cells that arise from the initial transformed cell will contain plasmids with the same inserted sequence of DNA (). The inserted DNA is replicated along with the rest of the plasmid DNA and segregates to daughter cells as the colony grows. In this way, the initial fragment of DNA is replicated in the colony of cells into a large number of identical copies. Since all the cells in a colony arise from a single transformed parental cell, they constitute a clone of cells. The initial fragment of DNA inserted into the parental plasmid is referred to as cloned DNA, since it can be isolated from the clone of cells.

General procedure for cloning a DNA fragment in a plasmid vector. Although not indicated by color, the plasmid contains a replication origin and ampicillin-resistance gene. Uptake of plasmids by E. coli cells is stimulated by high concentrations of CaCl (more...)

DNA cloning allows fragments of DNA with a particular nucleotide sequence to be isolated from a complex mixture of fragments with many different sequences. As a simple example, assume you have a solution containing four different types of DNA fragments, each with a unique sequence (). Each fragment type is individually inserted into a plasmid vector. The resulting mixture of recombinant plasmids is incubated with E. coli cells under conditions that facilitate transformation; the cells then are cultured on antibiotic selective plates. Since each colony that develops arose from a single cell that took up a single plasmid, all the cells in a colony harbor the identical type of plasmid characterized by the DNA fragment inserted into it. As a result, copies of the DNA fragments in the initial mixture are isolated from one another in the separate bacterial colonies. DNA cloning thus is a powerful, yet simple method for purifying a particular DNA fragment from a complex mixture of fragments and producing large numbers of the fragment of interest.

Isolation of DNA fragments from a mixture by cloning in a plasmid vector. Four distinct DNA fragments, depicted in different colors, are inserted into plasmid cloning vectors, yielding a mixture of recombinant plasmids each containing a single DNA fragment. (more...)

To clone specific DNA fragments in a plasmid vector, as just described, or in other vectors discussed in later sections, the fragments must be produced and then inserted into the vector DNA. As noted in the introduction, restriction enzymes and DNA ligases are utilized to produce such recombinant DNA molecules.

Restriction enzymes are bacterial enzymes that recognize specific 4- to 8-bp sequences, called restriction sites, and then cleave both DNA strands at this site. Since these enzymes cleave DNA within the molecule, they are also called restriction endonucleases to distinguish them from exonucleases, which digest nucleic acids from an end. Many restriction sites, like the EcoRI site shown in , are short inverted repeat sequences; that is, the restriction-site sequence is the same on each DNA strand when read in the 53 direction. Because the DNA isolated from an individual organism has a specific sequence, restriction enzymes cut the DNA into a reproducible set of fragments called restriction fragments ().

Restriction-recognition sites are short DNA sequences recognized and cleaved by various restriction endonucleases. (a) EcoRI, a restriction enzyme from E. coli, makes staggered cuts at the specific 6-bp inverted repeat sequence shown. This cleavage yields (more...)

Fragments produced by cleavage of the 36-kb DNA genome from adenovirus 2 (Ad2) by EcoRI and another restriction enzyme, HindIII from Haemophilus influenzae. Double-stranded DNA is represented by single black lines in this figure. Digestion of (more...)

The word restriction in the name of these enzymes refers to their function in the bacteria from which they are isolated: a restriction endonuclease destroys (restricts) incoming foreign DNA (e.g., bacteriophage DNA or DNA taken up during transformation) by cleaving it at all the restriction sites in the DNA. Another enzyme, called a modification enzyme, protects a bacteriums own DNA from cleavage by modifying it at or near each potential cleavage site. The modification enzyme adds a methyl group to one or two bases, usually within the restriction site. When a methyl group is present there, the restriction endonuclease is prevented from cutting the DNA (). Together with the restriction endonuclease, the methylating enzyme forms a restriction-modification system that protects the host DNA while it destroys foreign DNA. Restriction enzymes have been purified from several hundred different species of bacteria, allowing DNA molecules to be cut at a large number of different sequences corresponding to the recognition sites of these enzymes ().

Selected Restriction Endonucleases and Their Restriction-Site Sequences.

As illustrated in , EcoRI makes staggered cuts in the two DNA strands. Many other restriction enzymes make similar cuts, generating fragments that have a single-stranded tail at both ends. The tails on the fragments generated at a given restriction site are complementary to those on all other fragments generated by the same restriction enzyme. At room temperature, these single-stranded regions, often called sticky ends, can transiently base-pair with those on other DNA fragments generated with the same restriction enzyme, regardless of the source of the DNA. This base pairing of sticky ends permits DNA from widely differing species to be ligated, forming chimeric molecules.

During in vivo DNA replication, DNA ligase catalyzes formation of 35 phosphodiester bonds between the short fragments of the discontinuously synthesized DNA strand at a replication fork (see ). In recombinant DNA technology, purified DNA ligase is used to covalently join the ends of restriction fragments in vitro. This enzyme can catalyze the formation of a 35 phosphodiester bond between the 3-hydroxyl end of one restriction-fragment strand and the 5-phosphate end of another restriction-fragment strand during the time that the sticky ends are transiently base-paired (). When DNA ligase and ATP are added to a solution containing restriction fragments with sticky ends, the restriction fragments are covalently ligated together through the standard 35 phosphodiester bonds of DNA.

Ligation of restriction fragments with complementary sticky ends. In this example, EcoRI fragments from DNA I (left) are mixed with several different restriction fragments, including EcoRI fragments, produced from DNA II (right). The short DNA sequences (more...)

Some restriction enzymes, such as AluI and SmaI, cleave both DNA strands at the same point within the recognition site (see ). These restriction enzymes generate DNA restriction fragments with blunt (flush) ends in which all the nucleotides at the fragment ends are base-paired to nucleotides in the complementary strand. In addition to ligating complementary sticky ends, the DNA ligase from bacteriophage T4 can ligate any two blunt DNA ends. However, blunt-end ligation requires a higher DNA concentration than ligation of sticky ends.

Restriction enzymes to create fragments with sticky ends and DNA ligase to covalently link them allow foreign DNA to be inserted into plasmid vectors in vitro in a straightforward procedure. E. coli plasmid vectors can be constructed with a polylinker, a synthetic multiple-cloning-site sequence that contains one copy of several different restriction sites (). When such a vector is treated with a restriction enzyme that recognizes a recognition sequence in the polylinker, it is cut at that sequence, generating sticky ends. In the presence of DNA ligase, DNA fragments produced with the same restriction enzyme will be inserted into the plasmid (). The ratio of DNA fragments to be inserted to cut vectors and other reaction conditions are chosen to maximize the insertion of one restriction fragment per plasmid vector. The recombinant plasmids produced in in vitro ligation reactions then can be used to transform antibiotic-sensitive E. coli cells as shown in . All the cells in each antibiotic-resistant clone that remains after selection contain plasmids with the same inserted DNA fragment, but different clones carry different fragments.

Plasmid vectors containing a polylinker, or multiple-cloning-site sequence, commonly are used to produce recombinant plasmids carrying exogenous DNA fragments. (a) Sequence of a polylinker that includes one copy of the recognition site, indicated by brackets, (more...)

Advances in synthetic chemistry now permit the chemical synthesis of single-stranded DNA (ssDNA) molecules of any sequence up to about 100 nucleotides in length. Synthetic DNA has a number of applications in recombinant DNA technology. Complementary ssDNAs can be synthesized and hybridized to each other to form a dsDNA with sticky ends. Such completely synthetic dsDNAs can be cloned into plasmid vectors just as DNA restriction fragments prepared from living organisms are. For example, the 57-bp polylinker sequence shown in was chemically synthesized and then inserted into plasmid vectors to facilitate the cloning of fragments generated by different restriction enzymes. This example illustrates the use of synthetic DNAs to add convenient restriction sites where they otherwise do not occur. As described later in the chapter, synthetic DNAs are used in sequencing DNA and as probes to identify clones of interest. Synthetic DNAs also can be substituted for natural DNA sequences in cloned DNA to study the effects of specific mutations; this topic is examined in Chapter 8.

The technique for chemical synthesis of DNA oligonucleotides is outlined in . Note that chains grow in the 35 direction, opposite to the direction of DNA chain growth catalyzed by DNA polymerases. Once the chemistry for producing synthetic DNA was standardized, automated instruments were developed that allow researchers to program the synthesis of oligonucleotides of specific sequences up to about 100 nucleotides long.

Chemical synthesis of oligonucleotides by sequential addition of reactive nucleotide derivatives in the 35 direction. The first nucleotide (monomer 1) is bound to a glass support by its 3 hydroxyl; (more...)

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DNA Cloning with Plasmid Vectors - Molecular Cell Biology ...

Cloning What is cloning?

The term cloning describes a number of different processes that can be used to produce genetically identical copies of a biological entity. The copied material, which has the same genetic makeup as the original, is referred to as a clone.

Researchers have cloned a wide range of biological materials, including genes, cells, tissues and even entire organisms, such as a sheep.

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Yes. In nature, some plants and single-celled organisms, such as bacteria, produce genetically identical offspring through a process called asexual reproduction. In asexual reproduction, a new individual is generated from a copy of a single cell from the parent organism.

Natural clones, also known as identical twins, occur in humans and other mammals. These twins are produced when a fertilized egg splits, creating two or more embryos that carry almost identical DNA. Identical twins have nearly the same genetic makeup as each other, but they are genetically different from either parent.

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There are three different types of artificial cloning: gene cloning, reproductive cloning and therapeutic cloning.

Gene cloning produces copies of genes or segments of DNA. Reproductive cloning produces copies of whole animals. Therapeutic cloning produces embryonic stem cells for experiments aimed at creating tissues to replace injured or diseased tissues.

Gene cloning, also known as DNA cloning, is a very different process from reproductive and therapeutic cloning. Reproductive and therapeutic cloning share many of the same techniques, but are done for different purposes.

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Gene cloning is the most common type of cloning done by researchers at the National Human Genome Research Institute (NHGRI). NHGRI researchers have not cloned any mammals and NHGRI does not clone humans.

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Researchers routinely use cloning techniques to make copies of genes that they wish to study. The procedure consists of inserting a gene from one organism, often referred to as "foreign DNA," into the genetic material of a carrier called a vector. Examples of vectors include bacteria, yeast cells, viruses or plasmids, which are small DNA circles carried by bacteria. After the gene is inserted, the vector is placed in laboratory conditions that prompt it to multiply, resulting in the gene being copied many times over.

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In reproductive cloning, researchers remove a mature somatic cell, such as a skin cell, from an animal that they wish to copy. They then transfer the DNA of the donor animal's somatic cell into an egg cell, or oocyte, that has had its own DNA-containing nucleus removed.

Researchers can add the DNA from the somatic cell to the empty egg in two different ways. In the first method, they remove the DNA-containing nucleus of the somatic cell with a needle and inject it into the empty egg. In the second approach, they use an electrical current to fuse the entire somatic cell with the empty egg.

In both processes, the egg is allowed to develop into an early-stage embryo in the test-tube and then is implanted into the womb of an adult female animal.

ltimately, the adult female gives birth to an animal that has the same genetic make up as the animal that donated the somatic cell. This young animal is referred to as a clone. Reproductive cloning may require the use of a surrogate mother to allow development of the cloned embryo, as was the case for the most famous cloned organism, Dolly the sheep.

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Over the last 50 years, scientists have conducted cloning experiments in a wide range of animals using a variety of techniques. In 1979, researchers produced the first genetically identical mice by splitting mouse embryos in the test tube and then implanting the resulting embryos into the wombs of adult female mice. Shortly after that, researchers produced the first genetically identical cows, sheep and chickens by transferring the nucleus of a cell taken from an early embryo into an egg that had been emptied of its nucleus.

It was not until 1996, however, that researchers succeeded in cloning the first mammal from a mature (somatic) cell taken from an adult animal. After 276 attempts, Scottish researchers finally produced Dolly, the lamb from the udder cell of a 6-year-old sheep. Two years later, researchers in Japan cloned eight calves from a single cow, but only four survived.

Besides cattle and sheep, other mammals that have been cloned from somatic cells include: cat, deer, dog, horse, mule, ox, rabbit and rat. In addition, a rhesus monkey has been cloned by embryo splitting.

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Despite several highly publicized claims, human cloning still appears to be fiction. There currently is no solid scientific evidence that anyone has cloned human embryos.

In 1998, scientists in South Korea claimed to have successfully cloned a human embryo, but said the experiment was interrupted very early when the clone was just a group of four cells. In 2002, Clonaid, part of a religious group that believes humans were created by extraterrestrials, held a news conference to announce the birth of what it claimed to be the first cloned human, a girl named Eve. However, despite repeated requests by the research community and the news media, Clonaid never provided any evidence to confirm the existence of this clone or the other 12 human clones it purportedly created.

In 2004, a group led by Woo-Suk Hwang of Seoul National University in South Korea published a paper in the journal Science in which it claimed to have created a cloned human embryo in a test tube. However, an independent scientific committee later found no proof to support the claim and, in January 2006, Science announced that Hwang's paper had been retracted.

From a technical perspective, cloning humans and other primates is more difficult than in other mammals. One reason is that two proteins essential to cell division, known as spindle proteins, are located very close to the chromosomes in primate eggs. Consequently, removal of the egg's nucleus to make room for the donor nucleus also removes the spindle proteins, interfering with cell division. In other mammals, such as cats, rabbits and mice, the two spindle proteins are spread throughout the egg. So, removal of the egg's nucleus does not result in loss of spindle proteins. In addition, some dyes and the ultraviolet light used to remove the egg's nucleus can damage the primate cell and prevent it from growing.

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No. Clones do not always look identical. Although clones share the same genetic material, the environment also plays a big role in how an organism turns out.

For example, the first cat to be cloned, named Cc, is a female calico cat that looks very different from her mother. The explanation for the difference is that the color and pattern of the coats of cats cannot be attributed exclusively to genes. A biological phenomenon involving inactivation of the X chromosome (See sex chromosome) in every cell of the female cat (which has two X chromosomes) determines which coat color genes are switched off and which are switched on. The distribution of X inactivation, which seems to occur randomly, determines the appearance of the cat's coat.

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Reproductive cloning may enable researchers to make copies of animals with the potential benefits for the fields of medicine and agriculture.

For instance, the same Scottish researchers who cloned Dolly have cloned other sheep that have been genetically modified to produce milk that contains a human protein essential for blood clotting. The hope is that someday this protein can be purified from the milk and given to humans whose blood does not clot properly. Another possible use of cloned animals is for testing new drugs and treatment strategies. The great advantage of using cloned animals for drug testing is that they are all genetically identical, which means their responses to the drugs should be uniform rather than variable as seen in animals with different genetic make-ups.

After consulting with many independent scientists and experts in cloning, the U.S. Food and Drug Administration (FDA) decided in January 2008 that meat and milk from cloned animals, such as cattle, pigs and goats, are as safe as those from non-cloned animals. The FDA action means that researchers are now free to using cloning methods to make copies of animals with desirable agricultural traits, such as high milk production or lean meat. However, because cloning is still very expensive, it will likely take many years until food products from cloned animals actually appear in supermarkets.

Another application is to create clones to build populations of endangered, or possibly even extinct, species of animals. In 2001, researchers produced the first clone of an endangered species: a type of Asian ox known as a guar. Sadly, the baby guar, which had developed inside a surrogate cow mother, died just a few days after its birth. In 2003, another endangered type of ox, called the Banteg, was successfully cloned. Soon after, three African wildcats were cloned using frozen embryos as a source of DNA. Although some experts think cloning can save many species that would otherwise disappear, others argue that cloning produces a population of genetically identical individuals that lack the genetic variability necessary for species survival.

Some people also have expressed interest in having their deceased pets cloned in the hope of getting a similar animal to replace the dead one. But as shown by Cc the cloned cat, a clone may not turn out exactly like the original pet whose DNA was used to make the clone.

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Reproductive cloning is a very inefficient technique and most cloned animal embryos cannot develop into healthy individuals. For instance, Dolly was the only clone to be born live out of a total of 277 cloned embryos. This very low efficiency, combined with safety concerns, presents a serious obstacle to the application of reproductive cloning.

Researchers have observed some adverse health effects in sheep and other mammals that have been cloned. These include an increase in birth size and a variety of defects in vital organs, such as the liver, brain and heart. Other consequences include premature aging and problems with the immune system. Another potential problem centers on the relative age of the cloned cell's chromosomes. As cells go through their normal rounds of division, the tips of the chromosomes, called telomeres, shrink. Over time, the telomeres become so short that the cell can no longer divide and, consequently, the cell dies. This is part of the natural aging process that seems to happen in all cell types. As a consequence, clones created from a cell taken from an adult might have chromosomes that are already shorter than normal, which may condemn the clones' cells to a shorter life span. Indeed, Dolly, who was cloned from the cell of a 6-year-old sheep, had chromosomes that were shorter than those of other sheep her age. Dolly died when she was six years old, about half the average sheep's 12-year lifespan.

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Therapeutic cloning involves creating a cloned embryo for the sole purpose of producing embryonic stem cells with the same DNA as the donor cell. These stem cells can be used in experiments aimed at understanding disease and developing new treatments for disease. To date, there is no evidence that human embryos have been produced for therapeutic cloning.

The richest source of embryonic stem cells is tissue formed during the first five days after the egg has started to divide. At this stage of development, called the blastocyst, the embryo consists of a cluster of about 100 cells that can become any cell type. Stem cells are harvested from cloned embryos at this stage of development, resulting in destruction of the embryo while it is still in the test tube.

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Researchers hope to use embryonic stem cells, which have the unique ability to generate virtually all types of cells in an organism, to grow healthy tissues in the laboratory that can be used replace injured or diseased tissues. In addition, it may be possible to learn more about the molecular causes of disease by studying embryonic stem cell lines from cloned embryos derived from the cells of animals or humans with different diseases. Finally, differentiated tissues derived from ES cells are excellent tools to test new therapeutic drugs.

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Many researchers think it is worthwhile to explore the use of embryonic stem cells as a path for treating human diseases. However, some experts are concerned about the striking similarities between stem cells and cancer cells. Both cell types have the ability to proliferate indefinitely and some studies show that after 60 cycles of cell division, stem cells can accumulate mutations that could lead to cancer. Therefore, the relationship between stem cells and cancer cells needs to be more clearly understood if stem cells are to be used to treat human disease.

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Gene cloning is a carefully regulated technique that is largely accepted today and used routinely in many labs worldwide. However, both reproductive and therapeutic cloning raise important ethical issues, especially as related to the potential use of these techniques in humans.

Reproductive cloning would present the potential of creating a human that is genetically identical to another person who has previously existed or who still exists. This may conflict with long-standing religious and societal values about human dignity, possibly infringing upon principles of individual freedom, identity and autonomy. However, some argue that reproductive cloning could help sterile couples fulfill their dream of parenthood. Others see human cloning as a way to avoid passing on a deleterious gene that runs in the family without having to undergo embryo screening or embryo selection.

Therapeutic cloning, while offering the potential for treating humans suffering from disease or injury, would require the destruction of human embryos in the test tube. Consequently, opponents argue that using this technique to collect embryonic stem cells is wrong, regardless of whether such cells are used to benefit sick or injured people.

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Last Reviewed: May 11, 2016

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Cloning Fact Sheet