Due to the very low temperatures required, varying levels of stress are put on the DNA samples.  Spermatozoa , in particular, are stressed by temperature shock, osmotic stress, and oxidative stress with the latter being the most detrimental. [ 20 ]  When temperature shock occurs, the membrane is damaged through freezing and thawing of the sperm. Osmotic stress occurs when ice crystals form inside the nucleus during the freezing process, causing differing osmotic pressures within the cell. Oxidative stress is the result of too many reactive oxygen species (ROS), which is highly reactive and damaging to all parts of the cell. [ 20 ] [ 21 ]  Although these stressors are present within the cell, there are solutions to each. By introducing cholesterol to the samples, temperature shock can be reduced. The use of antifreeze proteins provides one solution for osmotic stress. Oxidative stress is the most difficult to combat because of the highly reactive components of ROS, but some measures like adding certain proteins to limit freeze-thaw damage and increase the survival rate of the DNA. [ 20 ]


A  gaur  that died of natural causes had some skin cells frozen and added to the San Diego Frozen Zoo. Eight years later, DNA from these cells was inserted into a domestic-cow egg to create an embryo (trans-species cloning), which was then implanted in a  domestic cow  ( Bos taurus ). On 8 January 2001, the gaur, named Noah, was born in  Sioux Center, Iowa . Noah was initially healthy, but the next day, he came down with clostridial enteritis, and died of  dysentery  within 48 hours of birth. This is not uncommon in uncloned animals, and the researchers did not think it was due to the cloning. [ 22 ]


The  banteng  was the second endangered species to be successfully  cloned , and the first clone to survive beyond infancy. [ 23 ] [ 24 ]  Scientists at  Advanced Cell Technology  in  Worcester, Massachusetts , extracted DNA from skin cells of a dead male banteng, that were preserved in San Diego 's Frozen Zoo facility, and transferred it into  eggs  from domestic banteng cows, a process called  somatic cell nuclear transfer . Thirty  embryos  were created and implanted in domestic banteng cows. Two were carried to term and delivered by  Caesarian section . The first was born on 1 April 2003, and the second two days later. The second was  euthanized , apparently suffering from large offspring syndrome (an  overgrowth disorder ), but the first survived and lived for seven years at the San Diego Zoo, where it died in April 2010 after it broke a leg and was euthanized. [ 25 ] [ 26 ]


In 2020, the first cloned  Przewalski's horse  was born, the result of a collaboration between  San Diego Zoo Global ,  ViaGen Equine  and  Revive & Restore . [ 27 ]  The  cloning  was carried out by  somatic cell nuclear transfer  (SCNT), whereby a viable  embryo  is created by transplanting the  DNA -containing  nucleus  of a  somatic cell  into an immature egg cell ( oocyte ) that has had its own nucleus removed, producing offspring genetically identical to the somatic cell donor. [ 28 ]  Since the oocyte used was from a domestic horse, this was an example of interspecies SCNT. [ 29 ]


The somatic cell donor was a Przewalski's horse stallion named Kuporovic, born in the UK in 1975, and relocated three years later to the US, where he died in 1998. Due to concerns over the loss of  genetic variation  in the captive Przewalski's horse population, and in anticipation of the development of new cloning techniques, tissue from the stallion was  cryopreserved  at the San Diego Zoo's Frozen Zoo. Breeding of this individual in the 1980s had already substantially increased the genetic diversity of the captive population, after he was discovered to have more unique  alleles  than any other horse living at the time, including otherwise-lost genetic material from two of the original captive founders. [ 27 ]  To produce the clone, frozen skin  fibroblasts  were thawed, and grown in  cell culture . [ 30 ]  An oocyte was collected from a domestic horse, and its nucleus replaced by a nucleus collected from a cultured Przewalski's horse fibroblast. The resulting embryo was induced to begin division and was cultured until it reached the  blastocyst  stage, then implanted into a domestic horse  surrogate  mare, [ 30 ]  which carried the embryo to term and delivered a foal with the Przewalski's horse DNA of the long-deceased stallion.


The cloned horse was named Kurt, after Dr.  Kurt Benirschke , a  geneticist  who developed the idea of cryopreserving genetic material from species considered to be endangered. His ideas led to the creation of the Frozen Zoo as a genetic library. [ 31 ]  There is a breeding herd in the  San Diego Zoo Safari Park . [ 32 ]  Once the foal matured, he was relocated to the breeding herd at the  San Diego Zoo Safari Park , [ 32 ]  so as to pass Kuporovic's genes into the larger captive Przewalski's horse population and increase the genetic variation of the species. In 2023, a second horse, named Ollie, was cloned from the same cell line. [ 27 ]


To help mitigate  inbreeding depression  for two endangered species, the  Black-footed ferret (Mustela nigripes) ,  Revive & Restore  facilitates on-going efforts to clone individuals from historic cell lines stored at the San Diego Zoo Wildlife Alliance Frozen Zoo. The program seeks to restore genetic variation lost from the living gene pool.


On December 10, 2020, the world's first cloned  black-footed ferret  was born. This ferret, named  Elizabeth Ann , marked the first time a U.S. endangered species was successfully cloned. [ 33 ] [ 34 ]


The cells of two 1980s wild-caught  black-footed ferrets  that never bred in captivity were preserved in the San Diego Wildlife Alliance Frozen Zoo. One of them was cloned to increase genetic diversity in this species in December 2020. More clones of both are planned. They will initially be bred separately from the non-cloned population. [ 35 ]


The  Pyrenean ibex  went extinct in 2000. In 2003 frozen cells from the last one (a female killed by a falling branch) were used to clone 208 embryos, of which 7 successfully implanted in goats, and one made it to term. That one ibex died of respiratory failure just after birth; quite possibly as a result of the cloning process, its lungs had not developed properly. There may not be enough individuals' cells preserved to create a breeding population. [ 36 ] [ 37 ]  Despite the death of the ibex, DNA analysis revealed that the offspring was a legitimate clone from its last living descendent. [ 38 ]


Over the years, concerns over population declines of the  northern white rhinoceros  ( Ceratotherium simum cottoni ) have increased with the increasing value of their horns to poachers. Specifically, the population has declined nearly seventy percent from 2011 to 2019. [ 39 ]  Processes like SCNT can help aid in conservation efforts towards the revival of their population. Researchers are looking towards induced pluripotent stem cells (iPSC), as they hold limitless possibilities. [ 40 ]  With the lack of natural mating occurring within the species due to the limited number of them, this sub-species provides researchers the opportunity for iPSC intervention. Other methods, including artificial insemination with fresh semen (AI), have been used successfully in another sub-species, the Southern White Rhinoceros ( Ceratotherium simum simum ). [ 41 ]  Frozen-thawed semen has been tested and has seen some successes, helping solve issues with reproduction of the species as a whole. [ 41 ]


Genetic Savings & Clone, Inc.  was a company headquartered in  Sausalito, California  that offered commercial  pet  gene banking and  cloning  services, between 2004 and 2006. The company was launched by billionaire  John Sperling , the founder of  University of Phoenix . Its  Chief Executive Officer  was Lou Hawthorne. [ 1 ]


The company was founded as a result of the efforts to clone Lou Hawthorne's favorite family dog, Missy. The  Missyplicity  project generated enough interest that Lou Hawthorne decided to build a company devoted to dog and cat cloning.


The company opened for business in February 2000, funded production of the first cloned cat,  CC , in 2001, and launched its pet cloning service in February 2004, operating a "petbank", to which pet owners could send  tissue  samples for later use in cloning. The company delivered the world's first commercially cloned cat,  Little Nicky , in December 2004.  Little Nicky was sold to a  Texas  woman for a reported  US$ 50,000. He was a genetic twin of "Nicky," a 17-year-old  Maine Coon  cat that had been kept as a pet. Musician  Liam Lynch 's cat was cloned after its death, presumably making him the first celebrity to own a cloned pet.


As well as their success in cloning cats, the company also made significant advances in dog cloning research, although the technology was not mature enough to sustain the business.  The company closed in 2006.  Letters to this effect were sent out to clients at the end of September 2006, informing them of this decision and offering to transfer any genetic material to another facility. [ 2 ]


The company spurred widespread debate regarding the ethics and morality of pet cloning especially in light of the fact that animals are  euthanized  by their owners every day. Though the topic lost currency with the closure of the company, divergent arguments about these issues can still be found on some web sites. [ 3 ]


The  GloFish  is a  patented  and  trademarked  brand of   fluorescently  colored  genetically modified aquarium fish . They have been created from several different species of fish:  zebrafish  were the first GloFish available in pet stores, and recently the  black tetra ,  tiger barb , [ 1 ]   rainbow shark ,  Siamese fighting fish ,  X-ray tetra , and most recently  bronze corydoras [ 2 ]  have been added to the lineup. They are sold in many colors, trademarked as "Starfire Red", "Moonrise Pink", "Sunburst Orange", "Electric Green", "Cosmic Blue", and "Galactic Purple", although not all species are available in all colors.  Although not originally developed for the ornamental fish trade, it is one of the first  genetically modified animals  to become publicly available. The rights to GloFish are owned by Spectrum Brands, Inc., which purchased GloFish from Yorktown Technologies, the original developer of GloFish, in May 2017.


The original  zebrafish  (or zebra danio,  Danio rerio ) is a native of rivers in  India  and  Bangladesh . It measures three centimeters long and has gold and dark blue stripes. In 1999, Dr. Zhiyuan Gong [ 3 ]  and his colleagues at the  National University of Singapore  were working with a gene that encodes the  green fluorescent protein  (GFP), originally extracted from a  jellyfish , that naturally produced bright green  fluorescence . They inserted the gene into a zebrafish embryo, allowing it to integrate into the zebrafish's  genome , which caused the fish to be brightly fluorescent under both natural white light and ultraviolet light. Their goal was to develop a fish that could detect  pollution  by selectively fluorescing in the presence of  environmental   toxins . The development of the constantly fluorescing fish was the first step in this process, and the National University of  Singapore  filed a patent application on this work. [ 4 ]  Shortly thereafter, his team developed a line of red fluorescent zebra fish by adding a gene from a sea  coral , and orange-yellow fluorescent zebra fish, by adding a variant of the jellyfish gene. Later, a team of researchers at the  National Taiwan University , headed by Professor Huai-Jen Tsai, succeeded in creating a  medaka  (rice fish) with a fluorescent green color, which, like the zebrafish, is a  model organism  used in biology.


The scientists from NUS and businessmen Alan Blake and Richard Crockett from Yorktown Technologies, L.P., a company in  Austin, Texas , met and a deal was signed whereby Yorktown obtained the worldwide rights to market the fluorescent zebrafish, which Yorktown subsequently branded as "GloFish". At around the same time, a separate deal was made between Taikong, the largest  aquarium  fish producer in Taiwan, and the Taiwanese researchers to market the green medaka in  Taiwan  under the name TK-1. In the spring of 2003, Taiwan became the first to authorize sales of a genetically modified organism as a pet. One hundred thousand fish were reportedly sold in less than a month at US$18.60 each. The fluorescent medaka are not GloFish, as they are not marketed by Yorktown Technologies, but instead by Taikong Corp under a different brand name.


GloFish were introduced to the  United States  market in late 2003 by Yorktown Technologies, after two years of research. The governmental environmental risk assessment was made by the U.S.  Food and Drug Administration  (FDA), which has jurisdiction over all genetically modified (GM) animals, including fluorescent zebra fish, since they consider the inserted gene to be a drug. The FDA determined in December 2003:


Because tropical aquarium fish are not used for food purposes, they pose no threat to the food supply. There is no evidence that these genetically engineered zebra danio fish pose any more threat to the environment than their unmodified counterparts which have long been widely sold in the United States. In the absence of a clear risk to the public health, the FDA finds no reason to regulate these particular fish. [ 5 ]


Marketing of the fish was met by protests from a  non-governmental organization  called the  Center for Food Safety . They were concerned that approval of the GloFish based only on a Food and Drug Administration risk assessment would create a precedent of inadequate scrutiny of biotech animals in general. [ citation needed ]  The group filed a lawsuit in US Federal District Court to block the sale of the GloFish. The lawsuit sought a court order stating that the sale of  transgenic  fish is subject to federal regulation beyond the FDA's charter, and as such should not be sold without more extensive approvals. In the opinion of Joseph Mendelson, the Center for Food Safety's legal director:


It's clear this sets a precedent for genetically engineered animals. It opens the dams to a whole host of nonfood genetically engineered organisms. That's unacceptable to us and runs counter to things the National Academy of Sciences and other scientific review boards have said, particularly when it comes to mobile GM organisms like fish and insects. [ 6 ]


The Center for Food Safety's suit was found to be without merit and dismissed on March 30, 2005. [ citation needed ]


In addition to the red zebrafish, Yorktown Technologies released green and orange-yellow versions of the zebrafish in mid-2006. In 2011, blue and purple zebrafish were released. These lines of fish incorporate genes from sea coral. [ 1 ]  In 2012, Yorktown Technologies introduced a green version of a GloFish derived from a different species of fish, the  black tetra . [ 1 ]  This was followed by a green version of a  tiger barb . In 2013, Yorktown Technologies introduced orange, pink, and purple Tetras, which made Tetras the first GloFish to be available in pink. This was followed in 2014 by the release of red and blue Tetras. The colors are trademarked as "Starfire Red", "Moonrise Pink", "Sunburst Orange", "Electric Green", "Cosmic Blue", and "Galactic Purple".


Other fish released include the GloFish shark, available in orange, green, and purple. Though these fish are not scientifically related to sharks, they are based on the albino rainbow shark. [ 7 ]  In February 2020, green GloFish bettas also known as Globettas were released, with three different variations. These variations include female, (young) male, and premium (adult) male.


Despite the speculation of aquarium enthusiasts that the eggs of the fluorescent fish were pressure treated to make them infertile, it has been found some GloFish are indeed fertile and will reproduce in a captive environment. [ 8 ]  However, the GloFish Fluorescent Fish License states "Intentional breeding and/or any sale, barter, or trade, of any offspring of GloFish fluorescent ornamental fish is strictly prohibited.". [ 9 ]


Sale or possession of GloFish was made illegal in  California  in 2003 due to a regulation that restricts genetically modified fish. The regulation was implemented before the marketing of GloFish, largely due to concern about a fast-growing biotech salmon. The regulations were lifted in 2015 due to a growing body of evidence and the findings of the Food and Drug Administration and the  Florida Department of Agriculture and Consumer Services . GloFish are now legal in California for importation and commercial sale. [ 10 ]


The import, sale and possession of these fish is not permitted within the European Union. On November 9, 2006, however, the Netherlands’ Ministry of Housing, Spatial Planning and the Environment (VROM) found 1,400 fluorescent fish, which were sold in various aquarium shops. [ 11 ]


In January 2009, the U.S. Food & Drug Administration formalized their recommendations for genetically engineered animals. [ 12 ]  These non-binding recommendations describe the way in which FDA regulates all GM animals, including GloFish. [ 13 ]


Research published in 2014 assessed the environmental safety associated with GloFish. One paper concluded that there is little risk of  invasiveness  into the environment. [ 14 ]  A second study concluded that there is no difference in risk between GloFish and wild-type danios. [ 15 ]


The sentiments of aquarium retailers towards the GloFish have been used as an indicator of the public's positive reaction to controversial agricultural and aesthetic biotechnologies. The practical reception of GloFish among fish retailers was found to be affected by multiple effects, including concerns over ethics, customer demand, and the high cost of stocking the fish. Some retailers opted not to stock the fish due to low trust in federal agencies to properly regulate the organisms. [ 16 ]


GloFish are more vulnerable to  predation  compared to the wild type, according to a study published in 2011. In experiments including habitat complexity,  transgenic  red fluorescent zebrafish were approximately twice as vulnerable as the wild type to predation by largemouth bass ( Micropterus salmoides ) and eastern mosquitofish ( Gambusia holbrooki ), two native predators that potentially resist  invasion  by introduced fish. [ 17 ]


According to Howard  et al.  2015, wild-type males had a significant advantage over GloFish when it came to mating. [ 18 ]  According to the mating trials that were analyzed in the study, wild-type males sired twice as much as the genetically modified fish due to their more aggressive nature. [ 18 ]  However, in Owen  et al.  2012 by the same group, female zebrafish  preferred  the GloFish rather than wild-type males. [ 18 ]


Horse cloning  is the process of obtaining a horse with  genes  identical to that of another horse, using an artificial  fertilization  technique. Interest in this technique began in the 1980s. The Haflinger foal  Prometea , the first living cloned horse, was obtained in 2003 in an Italian laboratory. Over the years, the technique has improved. It is mainly used on high-performance but castrated or infertile animals, for reproductive cloning. These horses are then used as breeding stock. Horse cloning is only mastered by a handful of laboratories worldwide, notably in France, Argentina, North America and China. The technique is limited by the fact that some differences remain between the original and its clone, due to the influence of  mitochondrial DNA .


Reproductive cloning of the  Pieraz  and  Quidam de Revel  horses began in 2005. The  International Federation for Equestrian Sports  (FEI by its acronym in French) decided to ban clones from competition in 2007, before authorizing them in 2012. A few clones are used in  equestrian sports , winning major titles such as the Argentine  polo  championship in 2013. Nevertheless, the number of cloned horses is growing every year. The practice is highly controversial, particularly for  bioethical reasons , since it involves a high failure rate on  embryos . It also raises questions about the management of horses'  genetic diversity , the future of the  horse breeding  profession, and the outbreak of new  genetic disorders  or fraud.


The horse is the seventh  species  to be cloned yet. [ 1 ]


Horse cloning has undergone a rapid qualitative and quantitative evolution. [ 2 ]  While Italian professor Cesare Galli believes that horse cloning has aroused less interest than that of other large mammals, [ 3 ]  other scientists believe that the high commercial value attained by some horses has created immediate interest, unlike in the case of less valuable agricultural animal species. [ 4 ]  Equine cloning owes much of its development to the Belgian stud farm of  Zangersheide , one of the pioneers of  artificial insemination  and  embryo transfer . According to Éric Palmer, a French biologist specializing in horse reproduction (who introduced  ultrasound  to mares and produced the first foal by  in vitro fertilization ), [ 5 ]  the way for the use of cloning was initiated in the 1980s by  veterinarian  surgeon Dr. Leo de Backer. He was in contact with some of the world's leading sports stables. According to Palmer, the ones that are interested are  Alwin Schockemöhle ,  Jan Tops , Thomas Fruhman,  John  and  Michael Whitaker ,  Willi Melliger , Jean-Claude Van Geensbergen and La Silla (in Mexico), among many others. [ 6 ]  The value of cloning high lineage horses was recognized as early as 1998, with the Westhusin study. [ 7 ]  Research to this end was publicly announced in 2001. [ 8 ]  That same year, with the support of  Genopole , Éric Palmer founded Cryozootech, a company dedicated to preserving the genes of horses with exceptional performance, with a view to future cloning. The horse is not the first large mammal to be cloned, as  Dolly  the sheep and other animals precede it, making it the seventh mammal to be cloned. [ 9 ]


The birth of three cloned mules in the United States on May 4, 2003, came just before that of the first horse. [ 10 ]  The first successful attempt to produce a viable clone was made by the Italian laboratory LTR-CIZ, which gave birth to  Prometea  on May 28, 2003, a  Haflinger  foal carried to term by her mother, whose genetic copy she is. [ 9 ]  Her birth was announced publicly on August 6, 2003. Born 36 kilogram after a natural delivery and a full-term pregnancy in Laboratory of Reproductive Technology,  Cremona ,  Italy , [ 11 ] [ full citation needed ]  At 2 months old, Prometea weighed 100 kg (220 lb) [ citation needed ]  The name "Prometea" is the  feminine  form of Prometeo (" Prometheus " in  Greek ). These scientists worked under the guidance of Professor Cesare Galli. [ 9 ]


Dr. Cesare Galli and others at the lab experimented with 841 reconstructed embryos; of the 14 viable embryos, four were implanted in surrogate mothers - only that of Prometea succeeded in being born. Prometea was born to her twin mother who her cloning cells originated from. [ 12 ]   Texas A&M University  was also undertaking a horse-cloning project when the Italian team first succeeded.


In 2002, LTR-CIZ merged with Cryozootech. In Italy, they produced the world's second cloned horse,  Pieraz-Cryozootech-Stallion . This is a purely commercial clone, aimed at obtaining a fertile genetic copy of a successful but castrated horse. According to  Bernard Debré , the birth of Pieraz-étalon heralded the commercial direction that equine cloning would later take. [ 13 ]  Prometea and  Pieraz  were obtained using the same method, that of Professor Galli. [ 10 ] [ 14 ]


Shortly afterwards, on 13 March 2005, Dr Katrin Hinrichs gave birth to Paris-Texas, a clone of  Quidam de Revel , in a laboratory at  Texas A&M University  in Texas (USA). The clone foal is also produced commercially for breeding purposes, at the request of  Quidam's  owner. The technique used is slightly different from that of the Italians. [ 15 ]  As a result, the number of clones produced has increased over the years. In 2009, the E.T. FRH clone became the first cloned  show jumping  horse authorized for breeding by a  studbook  (the  Zangersheide  studbook), while endurance champion  Pieraz's  clone entered its third breeding season. [ 16 ]  In Argentina, polo player  Adolfo Cambiaso  uses Crestview Genetics to clone his  polo ponies . At the end of 2010, a clone of his polo mare Cuartetera was sold at auction for a record  $ 800,000. [ 17 ] [ 18 ]  In May 2013, a non-clone foal was born for the first time from two parents cloned by  embryo transfer . [ 19 ] [ 20 ]  On 7 December 2013, a cloned  polo pony  won a major sporting competition for the first time. It was the Argentine polo championship. [ 21 ]  In 2018, equine cloning was widely used in Argentina's polo scene. [ 22 ]  The Argentine polo horse has become the most cloned animal in the world. [ 23 ]


Cloning research is often carried out in secret, due to poor public acceptance. [ 19 ]  Commercial cloning companies sometimes reveal the births of these horses, but the techniques employed remain mostly secret. [ 24 ]  According to the French national stud farm, the cloning technique used, known as "somatic" cloning, involves taking cells by  biopsy , usually from the breast of an adult animal.  Fibroblasts  are extracted and cultured in vitro until a sufficient number is obtained, then stored in  liquid nitrogen .  Oocytes  are harvested from a mare, either living or dead. The DNA is removed by  enucleation , then in vitro culture makes it suitable for receiving fibroblast DNA from the animal to be cloned. [ 25 ] [ 26 ]  Due to the high demand for mare oocytes, these are usually obtained from slaughterhouses. [ 3 ]


After a week or so of in vitro culture, the resulting  embryo  is implanted into the  uterus  of a carrier mare, using the  embryo transfer  technique. After eleven months'  gestation , the mare gives birth to the cloned foal. However, this type of gestation is much riskier than a conventional one. [ 25 ]


According to a Belgian researcher interviewed by  Le Vif , the failure rate is the main reason for opposition to cloning, for  bioethical reasons  due to the mortality of  embryos , fetuses and newborn  foals . [ 27 ]  This rate is high, but is gradually decreasing thanks to better-controlled techniques. Professor Galli obtained 15% viable embryos for his second clone,  Pieraz , compared with only 3% for  Prometea , [ 28 ]  the first horse, which required 328 attempts. [ 29 ]  The first  mule  trials, in 2003, involved 118 embryos, 13 of which produced a gestation, resulting in 3 live mules. [ 24 ]  To clone  Calvaro V  in 2006, Cryozootech used over 2,000  oocytes , which produced 22 embryos, only one of which was carried to term. [ 30 ]


Estimates of this rate vary from source to source. In 2012, according to a Belgian researcher, the average success rate for animal cloning was around 5%. [ 27 ]  Argentine researchers estimate that 6 or 7 embryos are needed out of 20 trials (in 2013). [ 31 ]  In 2010, according to a French source, around 2,500 mare oocytes had to be used to obtain a single viable foal. [ 26 ]  There are many abortions as well. Despite increased susceptibility to neonatal disease, a clone has the same life expectancy and robustness as a conventional horse. [ 32 ]  There is nothing to differentiate a clone from a conventionally bred horse. [ 4 ]


The cost of equine cloning varies between €200,000 [ 32 ]  and €300,000, [ 33 ]  depending on the source. In 2010, clones intended for sporting competitions represented just 22% of operations. [ 34 ]  Cloning is therefore mainly carried out in Europe for the purpose of breeding high-performance horses. A  gelding  can be copied to ensure its progeny. [ 35 ]  The same applies to a stallion that has become too old to reproduce, or to a mare whose number of foals is naturally limited. [ 25 ]  The use of cloning relies heavily on the belief that DNA is the most important factor in competition performance. [ 36 ]  Anne Ricard's study estimates that, in equestrian disciplines ( show jumping ,  dressage  and  endurance ) where geldings represent around 40% of competitors, the use of reproductive clones will enable a genetic improvement of 4% per generation. Once their fertility has been established, their semen is frozen as for any other stallion. [ 34 ] [ 37 ]


Effective cloning remains very marginal [ 34 ]  due to its cost. [ 31 ]  In the US and Argentina, requests for equine cloning come mainly from  polo  players (who allow their mares to play all the seasons) and  Arabian horse  breeders. [ 10 ]  Cloning can also be used to preserve rare breeds threatened with extinction, [ 38 ] [ 39 ]  but customers' motivations are essentially commercial. [ 34 ]  Nevertheless, the discovery of a perfectly preserved prehistoric foal in Siberia (in 2018) augurs well for cloning trials by Russian and Korean researchers, to resurrect extinct equine breeds or species. [ 40 ]


If the foal is the genetic copy of its donor, the question of the influence of the  mitochondria  that remain present in the recipient oocyte is still open. [ 41 ]  Mitochondria represent only 1 or 2% of the genome, but could influence the clone's sporting performance. [ 25 ]  They are more important in the case of a mare than a stallion, since the mare transmits her mitochondria during reproduction, unlike the stallion. [ 42 ]  Similarly, the cloned horse is not necessarily a perfect copy of the donor in terms of  phenotype  and character. The  horse markings  may vary, and the character, depending less on genetics than on the influence of the mother and upbringing, may also turn out to be very different. The technique also has its limits when it comes to breeding, as the pattern sought in horses evolves over time. There is therefore little point in cloning a horse clone. [ 32 ]  It also takes a long time to implement, and the number of specialized laboratories and companies is limited. The Kheiron company in Argentina, for example, estimates its waiting time at eighteen years, with demand far outstripping supply. [ 31 ]  The ban on clones in a large number of stud books and in certain competitions also limits interest. [ 26 ]


A few companies are known for their specialization in commercial equine cloning: ViaGen, Replica Farms, Crestview Genetics, Kheiron and Cryozootech. Competition between these laboratories is fierce. [ 31 ]  French company Cryozootech is a pioneer in the field, having produced the first commercial clone in 2005. [ 34 ]  It has made the production of famous horse clones its specialty. [ 43 ]  ViaGen was originally based in  Texas  in the United States, but the laboratory moved to Canada after the last American  slaughterhouses  closed in 2007, to source mare  oocytes . [ 44 ]  Kheiron was set up in Argentina in 2009 with a team of eight people. Equine cloning has developed strongly in this country, thanks in particular to demand from  polo  players, the profusion of mare oocytes available for research (the country exports a lot of  horse meat , and has numerous slaughterhouses supplying oocytes) and the easy breeding conditions in the  Pampas . [ 31 ]  In 2012, Argentina was estimated to be the country producing the most horse clones in the world. [ 45 ]  In  Texas , more than 900 clones were born between the creation of the first laboratory and 2014. [ 31 ]  In 2019, equine cloning companies are expected to open in China. [ 46 ]


Canada since 2007 [ 44 ]


Argentina


Canada


The use of clones for the genetic improvement of horses' sporting performance is recognized, including by veterinarians, although skepticism remains high among some professionals. [ 2 ]  According to Éric Palmer, acceptance of horse cloning is growing, and attitudes are changing, in the same way as the gradual acceptance of  in vitro fertilization  and  artificial insemination  techniques in horses. [ 32 ]  Fears of the birth of malformed or monstrous animals diminished when clone owners realized that their animals were in good health. [ 52 ]


In 2007, the  International Federation for Equestrian Sports  ruled that cloned horses should be banned from the official competitions it organizes, believing that opening up participation to clones would be unjust and unfair to the competition. It revised its opinion in July 2012. Horse clones are now allowed in all FEI competitions. [ 53 ]  This reversal is seen as an important sign of recognition of the usefulness of clones in sport horse breeding. [ 37 ]


In the United States, the National Cutting Horse Association and the National Barrel Horse Association allow clones in  cutting  and  barrel racing  competitions. [ 34 ]  The  American Quarter Horse Association  was taken to court by owners and riders of cloned horses in 2012, for refusing to allow these horses to take part in official breed competitions. [ 21 ] [ 54 ]  The initial ruling ordered the association to amend its bylaws to allow clones to compete. [ 55 ]


Sporting training of the Levisto Alpha Z clone makes it possible, even probable, for a clone horse to win an Olympic title in the future. [ 56 ]


Clones are generally not entered in the stud books of their respective breeds. The American  Jockey Club  refuses to allow cloned horses to race. In France, clones are also banned from trotting and galloping races. [ 21 ]  Several European  sport horse  and  warmblood  studbooks accept clones:  Zangersheide ; Anglo-European (AES);  Irish Sport Horse  (ISH);  Dutch Warmblood  (KWPN);  Belgian Warmblood  (BWP); and  Holsteiner . [ 25 ] [ 57 ] [ 58 ]  France's  national stud farms  advise against banning clones from the various studbooks, arguing that this will ultimately drive the best gene pool abroad. [ 34 ]


The existence of clones is not always made public, due to the poor reception they receive. [ 10 ]  Although in Belgium, Isabelle Donnay believes that commercial cloning of horses has not been very successful, [ 27 ]  on a global scale, their numbers have clearly increased over time. Equidia Life's 2013 survey describes the practice as "booming". [ 19 ]  In winter 2010, 56 clones were counted worldwide, produced by laboratories in Europe, the United States and South America. Americans clone more mares than Europeans. [ 34 ]  Between 2006 and 2011, at least 20  American Quarter Horses  were cloned. [ 59 ]  In 2014, there were an estimated 900 clones in the state of  Texas . [ 10 ]  In Europe, the Belgian  Zangersheide  stable regularly uses this technique, with four horses cloned between 2006 and 2013. [ 33 ]  The stallion Salute, one of Smart Little Lena's clones, was exported to Australia in 2010 for breeding. [ 44 ]


Dr. Éric Palmer, Cryozootech, France


Dr.  Éric Palmer, Cryozootech, France


Poetin II Z ( Zangersheide )


Murka's Gem


Gem Twist Alpha Z ( Zangersheide )


July 2012


2008 (success)


Ratina Z Beta ( Zangersheide )


Ratina Z Gamma ( Zangersheide )


South America's first cloned horse


Jazz Clone 2


Cruising Encore


As Cold As Ice Beta


Cocaine Beta Z


Chilli Morning III (Trey)


Chilli Morning IV (Quattro)


Nintendo 64


According to various surveys, including one carried out by Cheval Savoir in 2009, horse cloning is generally poorly accepted by riders and horse professionals. They believe it introduces unfair competition to "normal" horse breeders, while constituting a highly lucrative and  ethically  unacceptable activity. For French scientist Éric Palmer, the technique is  demonized  due to misunderstandings. [ 32 ]  The  American Quarter Horse Association  has stated that "[...] clones have no parents, cloning is not breeding. It's just photocopies of the same horse", pointing to its low success rate and the risk of as yet unknown  genetic disorder  developing. [ 88 ]  The  Jockey Club  was also strongly opposed. [ 37 ]  Dr. Thomas Reed, who owns the private stud Morningside in Ireland, is also publicly opposed to cloning after the accidental death of his stallion  Hickstead  in competition at the end of 2011. [ 51 ]


In 2015, the  European Union  voted to ban the cloning of farm animals (cattle, pigs, sheep, goats, and horses), and the sale of cloned livestock, their offspring, and products derived from them, such as meat and milk. The ban excluded cloning for research, and for the conservation of rare breeds and endangered species. [ 89 ] [ 90 ]  However, no law was passed after the vote. As of 2024, horse cloning continues to be legal in the EU, with the  Zangersheide  registry in  Belgium  offering three cloned stallions for breeding. [ 91 ]


Horse cloning, like that of other animal species, raises bioethical issues, since it involves a high mortality rate of  embryos ,  fetuses  and young foals. The Swiss National Stud's ethics study reports "massive loss during gestation", with less than 1% of oocytes obtained resulting in a live foal. Furthermore, foals born from cloning suffer from frequent health problems. An American study looked at 14 clones born between 2004 and 2008. [ 92 ]  Six (43%) were normal, while the other eight suffered from neonatal disorders, umbilical problems and limb deformities. [ 92 ]  There are a large number of stillborn foals, deaths in the first few days after birth, immune deficiencies, and muscle and bone deformities. Problems at foaling are common for both carrier mare and foal, with  cesarean section  being a common option. If foals survive their postnatal period, they do not appear to be more susceptible to disease thereafter. The question of their  longevity  remains unknown, as the first clones are still too young to draw any statistics. [ 93 ]


In the UK, researcher William (Twink) Allen was refused permission to continue his cloning trials in 2004 for these ethical reasons, as the cloned animals could present malformations, anomalies and diseases, according to the British authorities. [ 94 ]  Dr. Natasha Lane, of the  Society for the Prevention of Cruelty to Animals  (SPCA by its acronym in French), said it was not acceptable to clone animals by sacrificing embryos "just to get a gold medal". [ 95 ]  Allen spoke out against the decision, saying that the British government had "caved in to the  animal protection  lobby". [ 28 ]


Although horses are not threatened with extinction or other major problem now, cloning may create less genetic diversity among horses by using these horses to breed.  This increases the life time of one breeding set of genetics resulting in less variability in a population. In conservation biology, there are concerns related to the lack of genetic diversity that allows for continuation of the species through genetic variation. [ 96 ]


On 8 June 2005, a number of French farmers belonging to the Confédération Paysanne demonstrated in front of the  Genopole  d' Évry , Cryozootech's headquarters, to denounce the "seizure of living matter" and a future loss of  genetic diversity , arguing that the development of cloning will eventually lead to the disappearance of the  breeding  profession. [ 10 ]  A number of specialists warn against the widespread use of cloning, believing that it will seriously damage the equine breeding industry, particularly in equestrian sport, by reducing demand for naturally-born foals. Cloning would also drastically reduce genetic diversity, as the same genes "could be reproduced over and over again". [ 97 ] [ 98 ]


One fear that has arisen with cloning is that of new forms of fraud. In studbooks that refuse to accept clones, particularly the  Thoroughbred  studbook, it would be possible to pass off a horse cloned from a champion as another animal by falsifying its identification documents. [ 97 ]


The  Human Fertilisation and Embryology Act 1990  (c. 37) is an Act of the  Parliament of the United Kingdom . It created the  Human Fertilisation and Embryology Authority  which is in charge of human  embryo  research, along with monitoring and licensing fertility clinics in the United Kingdom. [ 1 ]


The Authority is composed of a chairman, a deputy chairman, and however many members are appointed by the UK Secretary of State. They are in charge of reviewing information about human embryos and subsequent development, provision of treatment services, and activities governed by the Act of 1990. [ 1 ]  The Authority also offers information and advice to people seeking treatment, and to those who have donated  gametes  or embryos for purposes or activities covered in the Act of 1990. Some of the subjects under the Human Fertilisation and Embryology Act of 1990 are prohibitions in connection with gametes, embryos, and  germ cells . [ 2 ]


The Act also addresses licensing conditions, code of practice, and procedure of approval involving human embryos. [ 3 ]  This only concerns human embryos which have reached the two cell  zygote  stage, at which they are considered "fertilised" in the act. [ 2 ]  It also governs the keeping and using of human embryos, but only outside a woman's body. The act contains amendments to UK law regarding termination of pregnancy, surrogacy and parental rights. [ 2 ]


The Human Fertilization and Embryology Act 1990 regulates ex-vivo human embryo creation and the research involving them. This act established the  Human Fertilisation and Embryology Authority  (HFEA) to regulate treatment and research in the UK involving human embryos.  In 2001, an extension of the Act legalized embryo research for the purposes of "increasing knowledge about the development of embryos," "increasing knowledge about serious disease," and "enabling any such knowledge to be applied in developing treatments for serious disease." The HFEA grants licenses and research permission for up to three years, based on approval of five steps by the Research License Committee. [ 4 ]


HFEA policies are reviewed by specialists in the field regularly. After research and literature are reviewed, and open public meetings are held, the summarized information is presented to the Human Fertilisation Embryology Authority. [ 4 ]


Donors must meet certain criteria in order to be eligible for sperm, egg, or embryo donation. The donor can donate for research purposes or fertility treatment. Donors should find a HFEA licensed clinic, or can go through the  National Gamete Donation Trust . [ 5 ]


The HFEA is carrying out a detailed review to determine the best way to reduce the risk of multiple pregnancies with  in vitro fertilization  (IVF). For example,  Nadya Suleman  (or "Octomom") is publicly known for giving birth to  octuplets  after IVF treatment.


This policy allows for the use of techniques which alter the mitochondrial DNA of the egg or an embryo used in IVF, to prevent serious mitochondrial diseases from being inherited.


The policies reviewed by HFEA cover everything from human reproductive cloning to the creation of human-animal hybrids, and include subjects such as ethics with scientific and social significance.


Sperm, eggs and embryos received in the donation process are currently tested for many medical conditions, and also quarantined for six months to reduce the risk of complications to the mother and child. Other than a screening for genetic disorders, donors are tested for  HIV ,  hepatitis B , and  hepatitis C . [ 6 ]


Embryos must be donated by a woman between the ages of 18 and 35 years old, who has also undergone a medical screening and given informed consent (which can be revoked at any point up until the embryo is used). [ 6 ]


"Welfare of the Child" review (multiple pregnancy), for people seeking IVF treatment. While there is always a risk of having a multiples pregnancy after receiving IVF treatment, HFEA is reviewing policies which will reduce this dangerous possibility. No more than two eggs or embryos can be legally implanted in a woman in an IVF treatment. There is a 25% success rate of this procedure per treatment cycle. [ 6 ]


Includes safety procedure regulations at fertility clinics; includes safe  cryopreservation  of eggs and embryos. Eggs and embryos are stored for ten years after the initial treatment. If the patient decides not to pursue another pregnancy, the eggs and embryos can be donated for research or to another couple for fertility treatments. [ 6 ]


In donor-assisted conception, the donor may not receive any monetary compensation (in the UK), although they may have related expenses covered. [ 7 ]


Sperm, eggs and embryos are stored in  liquid nitrogen  using cryopreservation (defined as the freezing of cells or whole tissues to sub-zero temperatures—the boiling point of liquid nitrogen). [ 8 ]
This method preserves living organisms in a state where they can be restored to how they were before freezing. [ 8 ]


A cryoprotective compound (a liquid called cryopreservation medium), along with carefully controlled cooling and warming cycles ensure that minimal damage is done to the cells. [ 9 ] [ 8 ]


However, the freezing process is still somewhat damaging. Therefore, men wishing to donate sperm or have it stored for future use must make six sperm deposits for every one child they wish to have, due to the 50% survival rate of the sperm in each deposit. [ 8 ]  The sperm is then put into straw shaped vials, and placed in a storage tank of either liquid nitrogen, or liquid nitrogen vapor. The sub-zero temperatures of the liquid generally range from -150 degrees Celsius, to -196 degrees Celsius.


According to HFEA, the storage period for both human gametes and embryos cannot exceed ten years. [ 9 ]  HFEA requires a full informed consent from each party that has any relation to the egg, gametes, or embryo, all of which must be stored in accordance with their consents. [ 9 ]


Exceptions to the informed consent of gamete storage:


The act states that it is legal to "take" gametes or accept those provided, and store them without a person's consent, if the person is considered incapable, or until they "acquire such capacity." [ 9 ]


However, under paragraphs 9 and 10 of HFEA 1990, a person's gametes cannot be legally stored in the UK after their death. [ 9 ]


In July 1982 the Warnock Committee Inquiry was established. It was "to consider recent and potential developments in medicine and science related to human fertilisation and embryology; to consider what policies and safeguards should be applied, including consideration of the social, ethical, and legal implications of these developments; and to make recommendations." [ 10 ]


The Warnock Report was published on 18 July 1984. The report stated that a regulator was needed due to the 'special status' of embryos. [ 10 ]


In 1985 the Interim Licensing Authority was created. It was supposed to regulate work and research regarding human in vitro fertilisation until a permanent government legislation was passed. It remained the only authority until 1990.


The Unborn Children Protection Bill was also created in 1985. It was written by Enoch Powell and prohibited embryonic research. The Health Secretary would only have been allowed an embryo to be kept and implanted if it was for the sole purpose of assisting a named woman to bear a child. No other reason was allowed. This bill was not passed. It was reintroduced in 1986, where it again failed to pass. This was repeated again 1989. [ 10 ]


The  Surrogacy Arrangements Act 1985  was the first law that governed surrogacy arrangements. It criminalized commercial surrogacy arrangements. [ 10 ]


In 1987 the framework for human fertilisation and embryology was created. A white paper was published in regards to the recommendations of the Warnock Report. [ 10 ]


In 1990 the Human Fertilisation and Embryology Act 1990 was passed. The  Human Fertilisation and Embryology Authority , HFEA, officially started work August 1, 1991. [ 10 ]


The act covers several areas:


Within the act an embryo is defined as a live human embryo where fertilisation is complete, complete is defined as the appearance of a two cell zygote.


The act states that eggs, sperm, and embryo can only be stored for a finite amount of time in very specific conditions that are regulated by the  Human Fertilisation and Embryology Authority . [ 11 ]


Research on human embryos can only be performed for specifically defined purposes that must be considered 'necessary and desirable' by the  Human Fertilisation and Embryology Authority . Research can only be performed on an embryo for a maximum of fourteen days or until the primitive streak appears. The genetic composition of any cell within the embryo cannot be altered during the embryo's formation for research. [ 11 ]


The act defined several purposes: [ 3 ]


Section 37 [ 12 ]  of the Act amends the  Abortion Act 1967 . The section specifies and broadens the conditions where abortion is legal.


Women who consider abortion are referred to two doctors. Each doctor then advises her whether abortion is a suitable decision based on the conditions listed below. An abortion is granted only when the doctors reach a unanimous decision that the woman may terminate her pregnancy. An abortion that is performed without this decision or under any other circumstances is considered unlawful.


Abortion may be granted under one of the following circumstances: [ 12 ]


The registered medical practitioner that performs the abortion will continue to act in accordance with the  Infant Life (Preservation) Act 1929 . [ 12 ]


In 1991 the statutory storage period and special expeditions sections were revisited. Regulations were extended storage periods for eggs and sperm. Licensing rules for egg and sperm storage were also clarified. [ 10 ]


A Disclosure of Information Act was created in 1992. This allowed the  Human Fertilisation and Embryology Authority  to disclose information to others with the patient's consent. for example, information could be shared with their general practitioner.


The  Criminal Justice and Public Order Act 1994  added section 156. This prohibited the treatment of cells from aborted embryos. During the same year the Parental Orders regulations allowed parental orders to be made in surrogacy cases. [ 10 ]


In 1996 the permitted storage period for embryos was extended.


The  Human Fertilisation and Embryology (Deceased Fathers) Act 2003  amended section 28 in 2000. [ 13 ]


Sperm may be taken from a deceased male to fertilize an egg if the corresponding man and woman were:


In 2001 the Human Fertilisation and Embryology Regulations were added. These regulations extended the purposes that an embryo can be created for in regards to research. [ 11 ]


In addition, the  Human Reproductive Cloning Act 2001  was passed. This essential made human reproductive cloning illegal by outlawing the implantation of research embryos.


As of 2004 the Disclosure of Donor Information Regulations were formed. Any sperm or egg donors registered after April 1, 2005, were required to pass on name and last address given to the offspring. [ 10 ]  During this time Parliament began reviewing the Human Fertilisation and Embryology Act 1990. [ 14 ]


Licensing of all establishments handling gametes for treatment was required as of 2007 in the Quality and Safety Regulations.


In 2006 a white paper was published regarding a revised legislation for fertility. This led to the  Human Fertilisation and Embryology Act 2008 , HFE, being passed. This was a major review of fertility legislation, updating and amending the act of 1990. In 2009 the HFE act was passed. This is the current law in the UK. [ 10 ]


The  Human Fertilisation and Embryology Act 2008  (c. 22) is an  act  of the  Parliament of the United Kingdom . The Act constitutes a major review and update of the  Human Fertilisation and Embryology Act 1990 .


According to the  Department of Health , the Act's key provisions are: [ 2 ]


The Bill's discussion in Parliament did not permit time to debate whether it should extend abortion rights under the  Abortion Act 1967  to also cover  Northern Ireland . The 2008 Act does not alter the status quo. [ 3 ]


The Act also repealed and replaced the  Human Reproductive Cloning Act 2001 .


The  Human Fertilisation and Embryology Authority  ( HFEA ) is an executive  non-departmental public body  of the  Department of Health and Social Care  in the United Kingdom. It is a statutory body that regulates and inspects all clinics in the United Kingdom providing  in vitro  fertilisation  (IVF),  artificial insemination  and the storage of human  eggs ,  sperm  or  embryos . It also regulates human embryo research.


After the birth of  Louise Brown , the world's first IVF baby, in 1978, there was concern about the implications of this new technology. In 1982, the UK government formed a committee chaired by philosopher  Mary Warnock  to look into the issues and see what action needed to be taken.


Hundreds of interested individuals including doctors, scientists and organisations such as health, patient and parent organisations as well as religious groups gave evidence to the committee.


In the years following the Warnock report, [ 2 ]  proposals were brought forward by the government in the publication of a white paper  Human Fertilisation and Embryology: A Framework for Legislation  in 1987. The Human Fertilisation and Embryology Act 1990 [ 3 ]  was drafted taking the report into account. [ citation needed ]


The 1990 Act provided for the establishment of the Human Fertilisation and Embryology Authority (HFEA), an executive, non-departmental public body, the first statutory body of its type in the world.
The HFEA is the independent regulator for IVF treatment and human embryo research and came into effect on 1 August 1991. The 1990 Act ensured the regulation, through licensing, of:


The Act also requires the HFEA keep a database of every IVF treatment carried out since that date and a database relating to all cycles and use of donated  gametes  (egg and sperm).


In 2001, the Human Fertilisation and Embryology (Research Purposes) Regulations 2001/188 extended the purposes for which embryo research could be licensed to include "increasing knowledge about the development of embryos", "increasing knowledge about serious disease", and "enabling any such knowledge to be applied in developing treatments for serious disease".


This allows researchers to carry out embryonic stem cell research and therapeutic cloning providing that an HFEA Licence Committee considers the use of embryos necessary or desirable for one of these purposes of research.


The Human Reproductive Cloning Act 2001 was introduced to explicitly prohibit reproductive cloning in the UK, but it was repealed by the  Human Fertilisation and Embryology Act 2008 .


In 2004, the Human Fertilisation and Embryology Authority (Disclosure of Donor Information) Regulations 2004/1511, enabled donor-conceived children to access the identity of their sperm, egg or embryo donor upon reaching the age of 18.


The Regulations were implemented on 1 April 2005 and any donor who donated sperm, eggs or embryos from that date onwards is, by law, identifiable. Since that date, any person born as a result of donation is entitled to request and receive the donor's name and last known address, once they reach the age of 18.


The European Union Tissues and Cells Directives (EUTCD) introduced common safety and quality standards for human tissues and cells across the European Union (EU).


The purpose of the directives was to facilitate a safer and easier exchange of tissues and cells (including human eggs and sperm) between member states and to improve safety standards for European citizens.
The EUTCD was adopted by the Council of Ministers on 2 March 2004 and published in the Official Journal of the European Union on 7 April 2004. Member States were obliged to comply with its provisions from 7 April 2006.


In 2005, the House of Commons Science and Technology Select Committee published a report on Human Reproductive Technologies and the Law.


This inquiry investigated the legislative framework provided by the 1990 Act and challenges presented by technological advance and "recent changes in ethical and societal attitudes".


In light of the Committee's report, and legislative changes that had already been made, the Department of Health undertook a review of the 1990 Act. They then held a public consultation based on their review of the Act, and following this published a White Paper, Review of the Human Fertilisation and Embryology Act, within which Government presented its initial proposals to revise the legislation.


A Joint Committee of both houses scrutinised the Government's recommendations, and provided its views on what ought to be the final form of the Bill to be brought to parliament.


The Bill was finally brought to the House of Lords in November 2007, passing through the House of Commons through Spring and Autumn of 2008, and finally receiving Royal Assent on 13 November 2008.
The HFE Act 2008 updates the law to ensure it is fit for purpose in the 21st century. It is divided into three parts:


The main new elements of the Act are:


The current statutory functions of the HFEA, as a regulator under the HFE Acts 1990 and 2008 and other legislation include:


Multiple pregnancy  is the single biggest risk to patients and children born as a result of fertility treatment. Women undergoing IVF treatment are twenty times more likely to have a multiple birth than if they conceive naturally.


After carefully considering views from clinics, patients and professional bodies, the HFEA decided to set a maximum multiple birth rate that clinics should not exceed, which will be lowered each year. All clinics will have their own strategy setting out how they will lower the multiple birth rate in their clinic by identifying the patients for whom single embryo transfer is the most appropriate treatment. The HFEA aims to reduce multiple births from IVF treatment to 10% over a period of years.


Former Chairs include Professor  Lisa Jardine ,  Walter Merricks , Shirley Harrison,  Lord Harries ,  Dame Suzi Leather ,  Baroness Deech ,  Sir Colin Campbell  and Sally Cheshire.


Other notable former members include  Professor Emily Jackson  and  Margaret Auld , [ 10 ]  former  Chief Nursing Officer  for Scotland.


The  Human Reproductive Cloning Act 2001  (c. 23) was an  Act  of the  Parliament of the United Kingdom  "to prohibit the placing in a woman of a human embryo which has been created otherwise than by fertilisation". The act received  Royal Assent  on 4 December 2001.


On 14 January 2001, the  British government  passed The Human Fertilisation and Embryology (Research Purposes) Regulations 2001 [ 1 ]  to amend the  Human Fertilisation and Embryology Act 1990  by extending allowable reasons for embryo research to permit research around stem cells and cell nuclear replacement, thus allowing  therapeutic cloning . However, on 15 November 2001, a  pro-life  group won a  High Court  legal challenge, which struck down the regulation and effectively left all forms of cloning unregulated in the UK. Their hope was that Parliament would fill this gap by passing prohibitive legislation. [ 2 ] [ 3 ]  Parliament was quick to pass the Human Reproductive Cloning Act 2001 in order to explicitly prohibit reproductive cloning. The remaining gap with regard to therapeutic cloning was closed when the appeals courts reversed the previous decision of the High Court. [ 4 ]


The act was repealed and replaced by the  Human Fertilisation and Embryology Act 2008 .


The  Hwang affair , [ 1 ]  or  Hwang scandal , [ 2 ]  or  Hwanggate , [ 3 ]  is a case of  scientific misconduct  and  ethical issues  surrounding a South Korean biologist,  Hwang Woo-suk , who claimed to have created the first  human embryonic stem cells  by  cloning  in 2004. [ 4 ] [ 5 ]  Hwang and his research team at the  Seoul National University  reported in the journal  Science  that they successfully developed a  somatic cell nuclear transfer  method with which they made the stem cells. In 2005, they published again in  Science  the successful cloning of 11 person-specific stem cells using 185 human eggs. [ 6 ]  The research was hailed as "a ground-breaking paper" in science. Hwang was elevated as "the pride of Korea", [ 7 ]  "national hero" [of Korea], [ 8 ]  and a "supreme scientist", [ 9 ]  to international praise and fame. [ 10 ] [ 11 ]  Recognitions and honours immediately followed, including South Korea's Presidential Award in Science and Technology, and  Time  magazine listing him among the "People Who Mattered 2004" [ 12 ]  and the most influential people "The 2004 Time 100". [ 13 ]


Suspicion and controversy arose in late 2005, when Hwang's collaborator,  Gerald Schatten  at the  University of Pittsburgh , came to know of the real source of  oocytes  (egg cells) used in the 2004 study. [ 14 ]  The eggs, reportedly from several voluntary donors, were from Hwang's two researchers, a fact which Hwang denied. The ethical issues made Schatten immediately break his ties with Hwang. In December 2005, a whistleblower informed  Science  of reuse of the same data. As the journal probed in, it was revealed that there was a lot more  data fabrication . [ 14 ]  The SNU immediately investigated the research work and found that both the 2004 and 2005 papers contained fabricated results. Hwang was compelled to resign from the university, [ 15 ]  and publicly confessed in January 2006 that the research papers were based on fabricated data. [ 14 ]   Science  immediately retracted the two papers. [ 16 ]


In 2009, the Seoul Central District Court convicted Hwang for  embezzlement  and  bioethical violations , sentencing him to a two-year imprisonment. [ 17 ]  The incident was then recorded as the scandal that "shook the world of science," [ 6 ]  and became "one of the most widely reported and universally disappointing cases of scientific fraud in history". [ 18 ]


Hwang Woo-suk was a professor of veterinary biotechnology at the Seoul National University and specialised in stem cell research. In 1993, he devised an  in vitro fertilisation  method with which he made the first assisted reproduction in cows. [ 19 ]  He rose to public notice in 1999 when he announced that he had successfully cloned a dairy cow, named  Yeongrong-i , and a few months later, a Korean cow,  Jin-i  (also reported as  Yin-i [ 8 ] ). The following year, he announced the preparation for cloning an endangered  Siberian tiger . [ 8 ]  It was a failed attempt, but nonetheless, his popularity in the Korean public escalated from wide media coverage. In 2002, he claimed creation of a genetically modified pig that could be used for human organ transplant. [ 6 ]  In 2003, he announced the successful cloning of a BSE ( bovine spongiform encephalopathy )-resistant cow. [ 20 ]  However, science sceptics raised concern over the absence of research papers for any of his claims. [ 19 ]


In 2004, Hwang announced the first complete cloning of a human embryo. [ 13 ] [ 21 ]  The research, published in the 12 March 2004 issue of  Science , was reported as "Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst." [ 22 ]  For its potential medical value to replace diseased and damaged cells, several scientists had previously tried to clone the human embryo, but in vain. [ 23 ]  Hwang's team had developed an improved method of somatic cell nuclear transfer using which they could transfer the nuclei of somatic (non-reproductive) cells into egg cells which had their nuclei removed. [ 24 ]  They used human egg cells and  cumulus cells , which are found in ovaries near the developing eggs and are known to be good source of nuclear transfer. After emptying an egg of its nucleus, they transferred the nucleus of the cumulus cell into it. The new egg cell divided normally and grew into a  blastocyst , an early embryo characterised by a hollow ball of cells. They isolated the outer  trophoblast  cells that are destined to become the placenta, discarding the inner cell mass that would form the placenta. When the trophoblast cells were cultured, they could divide and form different tissues, indicating that they were viable stem cells. [ 25 ]  The report concluded: "This study shows the feasibility of generating human ES [embryonic stem] cells from a somatic cell isolated from a living person." [ 22 ]  It was the first instance of cloning of adult human cells and human embryonic stem cells. [ 25 ]


Hwang publicly reported the research at the annual meeting of the  American Association for the Advancement of Science  (AAAS) in Seattle on 16 February 2004. [ 24 ]  He specified that they used 242 eggs from 16 unpaid volunteers, creating about 100 cells from which 30 embryos were developed. [ 26 ]  Since the embryos had adult DNA, the resulting stem cells became clones of the adult somatic cells. From the embryos, the stem cells were collected and grafted into mice in which they could grow into various body parts including muscle, bone, cartilage and connective tissues. [ 23 ]  The method ensured that immune rejection would be avoided so that it could be used for the treatment of genetic disorders, as Hwang explained: "Our approach opens the door for the use of these specially developed cells in transplantation medicine." [ 26 ]


Hwang's team reported another successful cloning of human cells in the 17 June 2005 issue of  Science , in this case, embryonic stem cells derived from skin cells. [ 27 ]  Their study claimed the creation of 11 different stem cell lines that were the exact match of DNA in people having a variety of diseases. The experiment used 185 eggs from 18 donors. [ 28 ]  The report explicitly stated that: "Patients voluntarily donated oocytes and somatic cells for therapeutic cloning research and relevant applications but not for reproductive cloning ... no financial reimbursement in any form was paid." [ 27 ]


When the 2004 research was announced, it was received with praise and admiration.  Donald Kennedy , editor-in-chief of  Science , remarked: "the generation of stem cells by somatic cell nuclear transfer methods involving the same individuals may hold promise for advances in transplantation technology that could help people affected by many devastating conditions." [ 26 ]   Michael S. Gazzaniga , neuroscientists and bioethicist at Dartmouth College, who had supported therapeutic cloning, commented it as "a major advance in biomedical cloning". [ 29 ]  American scientists took the news to criticise the weakness of US government in stem cell research and its prohibitive attitude. [ 26 ] [ 30 ]  As Helen Pearson reported in  Nature , the cloning accomplishment turned Asians into "scientific tigers". [ 24 ]


Time  reported that as a consequence of the achievement, "a medical and ethical door that had remained mostly closed was kicked wide open." [ 31 ]  Hwang and his colleague Shin Yong Moon were listed by  Time  at number 84 in its list of most influential people "The 2004 Time 100" in April 2004. [ 13 ]


The critical issue was on bioethics, as the method ultimately wasted many human embryos [ 32 ]  and could be used to create full human clones, as John T. Durkin argued in  Science : "the developmental events leading from fertilized ovum, to blastula, to embryo, to fetus, to fully formed adult constitute a continuum." [ 33 ]  Hwang claimed that the purpose was for medical applications only, and said in Seattle, "Reproductive cloning is strictly prohibited [in South Korea]." [ 30 ]  At the time, South Korea was developing its "Bioethics and Biosafety Act" to be enforced in 2005. The regulations proscribed human reproductive cloning and experimental fusion of human and animal embryos; even therapeutic cloning for diseases would require authorised approval. Based on this situation, Sang-yong Song of the Hanyang University, criticised Hwang for not waiting the forthcoming regulations and social consensus in Korea. [ 34 ]   Howard H. Kendler , psychologist at the University of California, had an unbiased viewpoint, commenting: "Although individuals will differ in their opinions, a democracy can decide whether the benefits of embryonic stem cell research outweigh any disadvantages. Science can assist in making this decision, but cannot dictate it." [ 35 ]


Hwang loved public attention and tried to establish a network of bureaucrats. To name his second cloned cow, he solicited President  Kim Dae-jung , who gave the name after a celebrated Korean geisha "Hwang Jin-i." [ 6 ]  As he announced the cloning of a  BSE -resistant cow in 2003, [ 20 ]  President  Roh Moo-hyun  visited his laboratory and was shown a dog cured from its injury using stem cell transfer, to which the president applauded, "this is not a science; this is a magic." [ 6 ]  From that point, Hwang received escalated research funding from the government that peaked in 2005 at around US$30 million. [ 3 ]  That year, Korean  Ministry of Science and Technology  officially honoured him as "Supreme Scientist" for the first time in Korea; the title carried US$15 million. [ 36 ]  The government set up the World Stem Cell Hub at  Seoul National University Hospital  on 19 October 2005, created and directed by Hwang. [ 37 ] [ 38 ]  On the day of opening,  3000  people registered for willingness to take stem cell therapy. [ 6 ]


In the 2004 report, Hwang's team prudently remarked that "we cannot completely exclude the possibility that the cells had a  parthenogenetic  origin." [ 22 ]  Reference to parthenogenesis, the ability of embryo development from egg cells without fertilisation, was relevant because it had been documented that stem cells are capable of such transformation. In 1984, an experiment demonstrated that a genetic mixture ( chimera ) of nuclei from the stem cells, one-cell-stage embryos of mouse could develop into full embryos. [ 39 ]  Researchers at  Advanced Cell Technology  (ACT) in Worcester, Massachusetts, further showed in 2002 that primate (in this case  crab-eating macaque ,  Macaca fascicularis ) stem cells grew into the blastocyst stage. [ 40 ]  The ACT subsequently announced that they created the human parthenogenetic cells, although the cells could not reach the blastocyst stage. [ 41 ]  In 2003, Gerald Schatten of the University of Pittsburgh and his team reported a failed attempt of stem cell cloning in  rhesus monkey , the cell divisions were always erratic and produced abnormal chromosomes. [ 42 ]  Schatten declared: "This reinforces the fact that the charlatans who claim to have cloned humans have never understood enough cell or developmental biology." [ 43 ]


In June 2023, Netflix released the documentary film  King of Clones , which covered Hwang Woo-suk and this affair.


In re Roslin Institute (Edinburgh) , 750 F.3d 1333 (Fed. Cir. 2014), [ 1 ]  is a 2014 decision of the  United States Court of Appeals for the Federal Circuit  rejecting a patent for a  cloned  sheep known as  "Dolly the Sheep" — the first mammal ever cloned from an adult somatic cell. [ 2 ]


Dolly was cloned in 1996 by  Ian Wilmut ,  Keith Campbell  and colleagues at the  Roslin Institute , part of the  University of Edinburgh  Scotland. [ 3 ]  The cloning method Campbell and Wilmut used to create Dolly constituted a breakthrough in scientific discovery. Known as  somatic cell nuclear transfer , this process involves removing the  nucleus  of a regular body cell and implanting that nucleus into an egg cell that has had its cell nucleus removed. A nucleus is the  organelle  that holds a cell's genetic material (its DNA). Campbell and Wilmut found that if the donor, somatic cell is arrested in the stage of the cell cycle where it is dormant and non-replicating (the quiescent phase) prior to nuclear transfer, the resulting fused cell will develop into an  embryo . The resulting cloned animal is an exact genetic replica of the adult mammal from which the somatic cell nucleus was taken. [ 2 ]


The patent application claims the cloned animal. Claim 155 is representative:


The US Patent and Trademark Office ( USPTO ) rejected the claims as patent ineligible under 35 U.S.C. § 101 "because it constituted a natural phenomenon that did not possess 'markedly different characteristics than any found in nature.' " A patent on the method was allowed, but it is not involved in this case. [ 4 ]


The Federal Circuit unanimously affirmed the PTO rejection of the claims in opinion by Judge  Timothy Dyk  joined by Judges  Kimberly Ann Moore  and  Evan Wallach .


It is "clear that naturally occurring organisms are not patentable." [ 5 ]  The patent in  Chakrabarty  claimed a genetically engineered bacterium that was capable of breaking down various components of crude oil. The patent applicant created this non-naturally occurring bacterium by adding four plasmids to a specific strain of bacteria. The Court held that the modified bacterium was patentable because it was "new" with "markedly different characteristics from any found in nature and one having the potential for significant utility." Accordingly, discoveries that possess "markedly different characteristics from any found in nature" are eligible for patent protection, but any existing organism or newly discovered plant found in the wild is not patentable. Similarly in  Association for Molecular Pathology v. Myriad Genetics, Inc. , [ 6 ]  the Court held that claims on two naturally occurring, isolated genes (BRCA1 and BRCA2), which can be examined to determine whether a person is likely to develop breast cancer, were patent ineligible invalid under § 101, because the BRCA genes themselves were unpatentable products of nature. [ 7 ]


It is not disputed that the donor sheep from which Dolly was cloned could not be patented, but Dolly is an exact copy of that unpatentable sheep. "Dolly's genetic identity to her donor parent renders her unpatentable." An exact copy of a preexisting animal in not patent eligible. The court added that related Supreme Court rulings "reinforce this conclusion":


For example, Supreme Court decisions regarding the preemptive force of federal patent law confirm that individuals are free to copy any unpatentable article, such as a live farm animal, so long as they do not infringe a patented method of copying.  Sears, Roebuck & Co. v. Stiffel Co.  clarified that a state may not "prohibit the copying of [an] article itself or award damages for such copying" when that article is ineligible for patent protection. [ 8 ]  In  Sears , the question was whether the defendant, Sears Roebuck & Co., could be held liable under state law for copying a lamp design whose patent protection had expired. The Court explained that "when the patent expires the monopoly created by it expires, too, and the right to make the article—including the right to make it in precisely the shape it carried when patented—passes to the public." The Court further clarified that "[a]n unpatentable article, like an article on which the patent has expired, is in the public domain and may be made and sold by whoever chooses to do so." Roslin's claimed clones are exact genetic copies of patent ineligible subject matter. Accordingly, they are not eligible for patent protection. [ 9 ]


Roslin argued that "environmental factors" lead to differences in shape, size, color, and behavior, that result from aging and the interaction of the animal with its environment. But Roslin acknowledged that any differences came about or were produced "quite independently of any effort of the patentee." As in the  Funk  case: "Their qualities are the work of nature. Those qualities are of course not patentable. For patents cannot issue for the discovery of the phenomena of nature." Roslin also argued that its clones are distinguishable from their original donor mammals because of differences in  mitochondrial DNA , which originates from the donor egg rather than the donor nucleus. But the claims do not describe clones that have markedly different characteristics from the donor animals of which they are copies. Finally, Roslin argued that its clones are patent eligible because "they are time-delayed versions of their donor mammals, and therefore different from their original mammals," but that is always true of any copy of an original. [ 10 ]


● Professor Dan Burk finds that "the  Roslin  opinion is hardly a model of coherent judicial reasoning, either on its own terms or with regard to the Supreme Court's subject matter jurisprudence to that point." He insists that Dolly the cloned sheep was not something found in nature, because "genetically identical mammals are not what one finds in the wild." Rather, "mammals such as sheep propagate via sexual recombination which typically renders them  not  genetically identical." Furthermore, Dolly was born as an old sheep:


By virtue of inheriting a mature set of somatic cell chromosomes, rather than the freshly recombined set of germ-line chromosomes that would accompany natural conception, Dolly began life with shortened  telomeres . Thus, Dolly was in a genetic sense 'born old' and lived a shortened life as a result.


Burk argues that the proper test of patent eligibility for such a product as Dolly the sheep is whether the claim preempts field of the described subject matter, so that "fundamental concepts and materials, on which all inventors must draw, [are] caught up in patent claims." He says that the Roslin patent would not "capture fundamental or basic science on which future invention will depend, or if it does so, there is no indication in the  Roslin  opinion that this informed the analysis of patentable subject matter." According to Burk, the patent would not be preemptive because its covers only sheep or other mammals "produced by the cloning process, a limitation that constrains the patent to the specific and novel implementation disclosed by the applicant." [ 11 ]


● Gene Quinn in  IP Watchdog  deplores the  Roslin  decision:


Another nail in the coffin of innovation and a functioning patent system all because decision makers don't have enough guts to state the obvious. Being able to create something that is identical to what nature creates is an extraordinary achievement that should be celebrated, should be fostered and incentivized, and should be awarded with a patent. [ 12 ]


● Student author Miriam Swedlow argues that  Roslin  would not prevent the patenting of extinct animals, such as the dodo, passenger pigeon, or wooly mammoth, because the cloned animal would not be identical to a natural animal. Like the cDNA in  Association for Molecular Pathology v. Myriad Genetics, Inc. , [ 6 ]  large portions of the genome of an extinct animal would need to be guessed at and extrapolated from non-extinct animals, because the DNA recoverable from fossils is decayed. Therefore, the "re-created animal will not be an exact genetic copy of an animal that already exists and will have different structural characteristics than the original species." They would therefore be comparable to artificial animals such as the  Oncomouse , which was patented. [ 13 ]  Moreover, the "fragile nature of extinct animal DNA allows for multiple avenues of differentiation" from the original, now-extinct animal, preempting none. [ 14 ]


The citations in this article are written in  Bluebook  style. Please see the  talk page  for more information.


Ligation  is the joining of two nucleotides, or two nucleic acid fragments, into a single polymeric chain through the action of an enzyme known as a  ligase . The reaction involves the formation of a  phosphodiester bond  between the 3'-hydroxyl terminus of one nucleotide and the 5'-phosphoryl terminus of another nucleotide, which results in the two nucleotides being linked consecutively on a single strand. Ligation works in fundamentally the same way for both DNA and RNA. A cofactor is generally involved in the reaction, usually  ATP  or  NAD + . Eukaryotic ligases belong to the ATP type, while the NAD+ type are found in bacteria (e.g.  E. coli ). [ 1 ]


Ligation occurs naturally as part of numerous cellular processes, including DNA replication, transcription, splicing, and recombination, and is also an essential laboratory procedure in  molecular cloning , whereby DNA fragments are joined to create  recombinant DNA  molecules (such as when a foreign DNA fragment is inserted into a  plasmid ). The discovery of DNA ligase dates back to 1967 and was an important event in the field of  molecular biology . [ 1 ]  Ligation in the laboratory is normally performed using  T4   DNA ligase . It is broadly used  in vitro  due to its capability of joining sticky-ended fragments as well as blunt-ended fragments. [ 2 ]  However, procedures for ligation without the use of standard DNA ligase are also popular. Human DNA ligase abnormalities have been linked to pathological disorders characterized by immunodeficiency, radiation sensitivity, and developmental problems. [ 3 ]


The mechanism of the ligation reaction was first elucidated in the laboratory of I. Robert Lehman. [ 4 ] [ 5 ]  Two fragments of DNA may be joined by  DNA ligase  which catalyzes the formation of a phosphodiester bond between the 3'-hydroxyl group (-OH) at one end of a strand of DNA and the 5'-phosphate group (-PO4) of another. In animals and  bacteriophages ,  ATP  is used as the energy source for the ligation, while in bacteria,  NAD +  is used. [ 6 ]


The DNA ligase first reacts with ATP or NAD + , forming a ligase-AMP intermediate with the  AMP  linked to the ε-amino group of lysine in the active site of the ligase via a phosphoramide bond. This  adenylyl  group is then transferred to the phosphate group at the 5' end of a DNA chain, forming a DNA-adenylate complex. Finally, a phosphodiester bond between the two DNA ends is formed via the nucleophilic attack of the 3'-hydroxyl at the end of a DNA strand on the activated 5′-phosphoryl group of another. [ 4 ]


A  nick in the DNA   (i.e. a break in one strand of a double-stranded DNA) can be repaired very efficiently by the ligase. However, a complicating feature of ligation conducted presents itself when ligating two separate DNA ends as the two ends need to come together before the ligation reaction can proceed. In the ligation reactions conducted in a laboratory, the ligation of DNA with  sticky or cohesive ends , the protruding strands of DNA may be annealed together already, therefore it is a relatively efficient process as it is equivalent to repairing two nicks in the DNA. However, in the ligation of  blunt-ends , which lack protruding ends for the DNA to anneal together, the process is dependent on random collision for the ends to align together before they can be ligated, and is consequently a much less efficient process. [ 7 ]   The DNA ligase from  E. coli  cannot ligate blunt-ended DNA except under conditions of molecular crowding, and it is therefore not normally used for ligation in the laboratory.   Instead the DNA ligase from  phage T4  is used as it can ligate blunt-ended DNA as well as single-stranded DNA. [ 8 ] [ 6 ]


In the laboratory, factors that affect an enzyme-mediated chemical reaction would naturally affect a ligation reaction, these include the concentration of enzyme and the reactants, the temperature of reaction and the length of time of incubation.  Ligation is complicated by the fact that the reaction can involve both inter- and intra-molecular reactions, but the desired ligation products in many ligation reactions (e.g. ligating a DNA fragment into a vector) needed first to be inter-molecular, i.e. between two different DNA molecules, followed by an intra-molecular reaction to seal and circularize the molecule. For efficient ligation, an additional annealing step is also necessary.


The three steps to form a new phosphodiester bond during ligation are: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. Mg(2+) is a cofactor for catalysis, therefore at high concentration of Mg(2+) the ligation efficiency is high. If the concentration of Mg(2+) is limited, the nick- sealing is the rate- limiting reaction of the process, and adenylylated DNA intermediate stays in the solution. Such adenylylation of the enzyme restrains the rebinding to the adenylylated DNA intermediate  comparison of an Achilles' heel of LIG1, and represents a risk if they are not fixed. [ 9 ]


The concentration of DNA can affect the rate of ligation, and whether the ligation is an inter-molecular or intra-molecular reaction.  Ligation involves joining up the ends of a DNA with other ends, however, each DNA fragment has two ends, and if the ends are compatible, a DNA molecule can circularize by joining its own ends.  At high DNA concentration, there is a greater chance of one end of a DNA molecule meeting the end of another DNA, thereby forming intermolecular ligation. At a lower DNA concentration, the chance that one end of a DNA molecule would meet the other end of the same molecule increases, therefore  intramolecular reaction  that circularizes the DNA is more likely.  The  transformation efficiency  of linear DNA is also much lower than circular DNA, and for the DNA to circularize, the DNA concentration should not be too high.  As a general rule, the total DNA concentration should be less than 10 μg/ml. [ 10 ]


The relative concentration of the DNA fragments, their length, as well as buffer conditions are also factors that can affect whether intermolecular or intramolecular reactions are favored.


The concentration of DNA can be artificially increased by adding condensing agents such as  cobalt hexamine  and biogenic  polyamines  such as  spermidine , or by using  crowding agents  such as  polyethylene glycol  (PEG) which also increase the effective concentration of enzymes. [ 11 ] [ 12 ]  Note however that additives such as cobalt hexamine can produce exclusively intermolecular reaction, [ 11 ]  resulting in linear  concatemers  rather than the circular DNA more suitable for  transformation  of plasmid DNA, and is therefore undesirable for plasmid ligation.  If it is necessary to use additives in plasmid ligation, the use of PEG is preferable as it can promote intramolecular as well as intermolecular ligation. [ 13 ]


As is usual for an enzyme, the higher the ligase concentration, the faster is the rate of ligation.  Blunt-end ligation is much less efficient than sticky end ligation, so a higher concentration of ligase is used in blunt-end ligations.  High DNA ligase concentration may be used in conjunction with PEG for a faster ligation, and they are the components often found in commercial kits designed for rapid ligation. [ 14 ] [ 15 ]


Two issues are involved when considering the temperature of a ligation reaction.  First, the optimum temperature for DNA ligase activity which is 37 ° C, and second, the  melting temperature  (T m ) of the DNA ends to be ligated.  The melting temperature is dependent on length and base composition of the DNA overhang—the greater the number of G and C, the higher the T m  since there are three hydrogen bonds formed between G-C base pair compared to two for A-T base pair—with some contribution from the stacking of the bases between fragments.  For the ligation reaction to proceed efficiently, the ends should be stably annealed, and in ligation experiments, the T m  of the DNA ends is generally much lower than 37 ° C.  The optimal temperature for ligating cohesive ends is therefore a compromise between the best temperature for DNA ligase activity and the T m  where the ends can associate. [ 16 ]   However, different restriction enzymes generates different ends, and the base composition of the ends produced by these enzymes may also differ, the melting temperature and therefore the optimal temperature can vary widely depending on the restriction enzymes used, and the optimum temperature for ligation may be between 4-15 ° C depending on the ends. [ 17 ] [ 18 ]  Ligations also often involve ligating ends generated from different restriction enzymes in the same reaction mixture, therefore it may not be practical to select optimal temperature for a particular ligation reaction and most protocols simply choose 12-16 ° C, room temperature, or 4 ° C. When conducting a ligation at  4 ° C, it is necessary to increase the time of ligation reaction, for example by leaving the ligation mixture overnight or longer in the fridge.


The  ionic strength  of the buffer used can affect the ligation.  The kinds of cations presence can also influence the ligation reaction, for example, excess amount of Na +  can cause the DNA to become more rigid and increase the likelihood of intermolecular ligation.  At high concentration of monovalent cation (>200 mM) ligation can also be almost completely inhibited. [ 19 ]  The standard buffer used for ligation is designed to minimize ionic effects. [ 20 ]


Restriction enzymes can generate a wide variety of ends in the DNA they digest, but in cloning experiments most commonly-used restriction enzymes  generate a 4-base single-stranded overhang called the sticky or cohesive end (exceptions include  Nde I  which generates a 2-base overhang, and those that generate blunt ends).   These sticky ends can anneal to other compatible ends and become ligated in a sticky-end (or cohesive end) ligation.   Eco RI  for example generates an AATT end, and since A and T have lower melting temperature than C and G, its melting temperature T m  is low at around 6 ° C. [ 21 ]  For most restriction enzymes, the overhangs generated have a T m  that is around 15 ° C. [ 20 ]   For practical purposes, sticky end ligations are performed at 12-16 ° C, or at room temperature, or alternatively at 4 ° C for a longer period.


For the insertion of a DNA fragment into a plasmid vector, it is preferable to use two different restriction enzymes to digest the DNA so that different ends are generated.  The two different ends can prevent the religation of the vector without any insert, and it also allows the fragment to be inserted in a directional manner.


When it is not possible to use two different sites, then the vector DNA may need to be dephosphorylated to avoid a high background of recircularized vector DNA with no insert. Without a phosphate group at the ends the vector cannot ligate to itself, but can be ligated to an insert with a phosphate group.  Dephosphorylation is commonly done using  calf-intestinal alkaline phosphatase  (CIAP) which removes the phosphate group from the 5′ end of digested DNA, but note that CIAP is not easy to inactivate and can interfere with ligation without an additional step to remove the CIAP, thereby resulting in failure of ligation. CIAP should not be used in excessive amount and should only be used when necessary.  Shrimp  alkaline phosphatase  (SAP) or Antarctic phosphatase (AP) are suitable alternative as they can be easily inactivated.


Blunt end ligation does not involve base-pairing of the protruding ends, so any blunt end may be ligated to another blunt end. Blunt ends may be generated by restriction enzymes such as  Sma I  and  Eco RV .  A major advantage of blunt-end cloning is that the desired insert does not require any restriction sites in its sequence as blunt-ends are usually generated in a  PCR , and the  PCR  generated blunt-ended DNA fragment may then be ligated into a blunt-ended vector generated from restriction digest.


Blunt-end  ligation, however, is much less efficient than sticky end ligation, typically the reaction is 100X slower than sticky-end ligation.  Since blunt-end does not have protruding ends, the ligation reaction depends on random collisions between the blunt-ends and is consequently much less efficient.  To compensate for the lower efficiency, the concentration of ligase used is higher than sticky end ligation (10x or more).  The concentration of DNA used in blunt-end ligation is also higher to increase the likelihood of collisions between ends, and longer incubation time may also be used for blunt-end ligations.


If both ends needed to be ligated into a vector are blunt-ended, then the vector needs to be dephosphorylated to minimize self-ligation.  This may be done using CIAP, but caution in its use is necessary as noted previously.  Since the vector has been dephosphorylated, and ligation requires the presence of a 5'-phosphate, the insert must be phosphorylated. Blunt-ended PCR product normally lacks a  5'-phosphate, therefore it needs to be phosphorylated by treatment with  T4 polynucleotide kinase . [ 22 ]


Blunt-end ligation is also reversibly inhibited by high concentration of ATP. [ 23 ]


PCR usually generates blunt-ended PCR products, but note that PCR using  Taq  polymerase  can add an extra adenine (A) to the 3' end of the PCR product.  This property may be exploited in  TA cloning  where the ends of the PCR product can anneal to the T end of a vector.   TA ligation  is therefore a form of sticky end ligation.  Blunt-ended vectors may be turned into vector for TA ligation with dideoxythymidine triphosphate (ddTTP) using terminal transferase.


For the cloning of an insert into a circular plasmid:


Sometimes ligation fail to produce the desired ligated products, and some of the possible reasons may be:


A number of commercially available DNA cloning kits use other methods of ligation that do not require the use of the usual DNA ligases.  These methods allow cloning to be done much more rapidly, as well as allowing for simpler transfer of cloned DNA insert to different  vectors .  These methods however require the use of specially designed  vectors  and components, and may lack flexibility.


Topoisomerase  can be used instead of ligase for ligation, and the cloning may be done more rapidly without the need for restriction digest of the vector or insert.  In this  TOPO cloning  method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating to both of the 5' ends of the PCR product, the topoisomerase is released and a circular vector is formed in the process. [ 28 ]


Another method of cloning without the use of ligase is by  DNA recombination , for example as used in the  Gateway cloning system . [ 29 ] [ 30 ]  The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination. [ 31 ]


Different types of ligases found in the studied organisms. For instance,  Nicotinamide adenine dinucleotide  (NAD+)-dependent ligase was found and isolated from bacterial organism, known as  E. coli  in second third of 20th century. Since then, this model has been widely used to study that DNA ligase family. Moreover, it is found in all bacteria. Examples of genes present in  E. coli  are LigA, which has essential functions affecting bacterial growth, and LigB. [ 32 ]


In mammals, including human 3 genes, namely Lig1, Lig3, Lig4 were identified. All eukaryotes contain multiple types of DNA ligases encoded by Lig genes. [ 33 ]  The smallest known eukaryotic ligase is  Chlorella  virus DNA ligase (ChVLig). It contains only 298 amino acids. When ChVLig is the only source of ligase in the cell, it can continue to support mitotic development, and nonhomologous end joining in budding yeasts. [ 34 ]  DNA Ligase I (Lig1) is accountable for Okazaki Fragments ligation. It is consist of 919 amino acids. In a complex process of DNA replication, DNA Ligase I recruited to replications machinery by protein interactions. Lig1 plays role in cell division in plants and yeasts. Knockout of the Lig1 gene is lethal in yeasts and some plants sprouts.  Nevertheless, studies of mouse embryogenesis have shown that until the middle of the growth process embryo developing without DNA ligase I. [ 35 ]


Enzymatic ligation has been used in various studies related to DNA nanostructures and lead to increase of  efficiency and stability. One of the methods is sealing of covalent DNA bond, namely phosphodiester bond and nicks. Reconstruction of those structures performed with assistance of ligation. For instance, T4 DNA ligase serve as a catalyst for sealing of a nick between 3 prime and 5 prime ends of DNA to make up strong  phosphodiester bond. Ligated structures have higher thermal stability values. [ 36 ]  T4 DNA ligase has many valuable properties such as already mentioned catalytic, but it is also responsible for sealing of the gaps between DNA strands, nick-closing activity, repair of the DNA damage, etc. [ 2 ]


In nanostructures architecture, molecular biology researches - ssDNA is an important application model. T4 DNA ligase used to cyclize short ssDNA fragments, but process is complicated by formation of secondary structures. On the other hand,  Taq  DNA ligase is a thermostable enzyme which can be applied at higher temperatures (45, 55 and 65 °C respectively). Since at these temperature range secondary structures less stable it is enhance cyclization efficiency of oligonucleotides. The kinetic, biological, and other parameters of nanostructures are influenced by presence of the secondary structures in DNA rings. However,  Taq  DNA ligation occur only when two complementary DNA strands are perfectly paired and have no gaps in between. [ 37 ]


Analysis of ligases activities, mutations, deficiencies widely used in drug design and biological researches to investigate diseases, pathologies developments and related rare acquired or inherited syndromes (e.g. DNA ligase IV syndrome). [ 38 ] [ 39 ] [ 40 ] [ 41 ]


The ligation procedure is prevalent in molecular biology cloning techniques, and it has been applied to define and characterize specific nucleotide sequences in the genome using Ligase Chain Reaction (LCR) or Polymerase Chain Reaction (PCR)-based amplification of ligated probes. [ 42 ]


Ligation may also serve as a  DNA analysis  method. [ 43 ]  Some techniques employ  rolling circle amplification . [ 43 ]  The most notable of these is described by Smolina  et al. , 2007 & Smolina  et al. , 2008 using  fluorescence in situ hybridization  and  peptide nucleic acids . [ 43 ]  They developed and employed this technique for analyses of bacterial chromosomes. [ 43 ]


Ligation-independent cloning  (LIC) is a form of  molecular cloning  that can be performed without the use of  restriction endonucleases  or  DNA ligase . The technique was developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. [ 1 ]  This allows genes to be cloned without the requirement of a restriction site for cloning that is absent from the gene insert. [ 2 ] [ 3 ] [ 4 ] [ clarification needed ]  LIC uses long complementary overhangs on the vector and the DNA insert to create a stable association between them.  [ 5 ]


Megan and Morag , two  domestic sheep , were the first  mammals  to have been successfully  cloned  from differentiated cells. [ 1 ]  They are not to be confused with  Dolly the sheep  which was the first animal to be successfully cloned from an adult somatic cell [ 2 ]  or  Polly the sheep  which was the first cloned and transgenic animal. [ 3 ]  Megan and Morag, like Dolly and Polly, were cloned at the  Roslin Institute  in  Edinburgh ,  Scotland  in 1995.