Human artificial chromosome  may be potentially useful as a gene transfer vectors for gene delivery into human cells, and a tool for expression studies and determining human chromosome function. It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector. [ 16 ] [ 17 ]


Viruses that infect plant and animal cells have also been manipulated to introduce foreign genes into plant and animal cells. The natural ability of viruses to adsorb to cells, introduce their DNA and replicate have made them ideal vehicles to transfer foreign DNA into eukaryotic cells in culture. A vector based on  Simian virus 40  (SV40) was used in first cloning experiment involving mammalian cells. A number of vectors based on other type of viruses like  Adenoviruses  and  Papilloma virus  have been used to clone genes in mammals. At present, retroviral vectors are popular for cloning genes in mammalian cells. In case of plants like  Cauliflower mosaic virus ,  Tobacco mosaic virus  and  Gemini viruses  have been used with limited success.


Many general purpose vectors such as  pUC19  usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for  E. coli   β-galactosidase , whose activity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate  X-gal  (5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based  lacZα  sequence of the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.


Commercial animal cloning  is the  cloning of animals  for  commercial  purposes, including  animal husbandry , medical research, competition camels and horses, pet cloning, and restoring populations of  endangered  and  extinct  animals. [ 1 ]  The practice was first demonstrated in 1996 with  Dolly the sheep .


Moving or copying all (or nearly all) genes from one  animal  to form a second, genetically nearly identical, animal is usually done using one of three methods: the Roslin technique, the Honolulu technique, or Artificial Twinning. [ 2 ]   The first two of these involve a process known as  somatic cell nuclear transfer . [ 3 ]  In this process, an  oocyte  is taken from a  surrogate  mother and undergoes enucleation, a process that removes the nucleus from inside the oocyte.  Somatic cells  are then taken from the animal that is being cloned, transferred into the blank oocyte in order to provide genetic material, and fused with the oocyte using an electrical current. The oocyte is then activated and re-inserted into the  surrogate  mother. The result is the formation of an animal that is almost genetically identical to the animal the somatic cells were taken from. [ 3 ] [ 4 ]  While somatic cell nuclear transfer was previously believed to only work using genetic material from somatic cells that were unfrozen or were frozen with  cryoprotectant  (to avoid cell damage caused by freezing), successful dog cloning in various breeds has now been shown using somatic cells from unprotected specimens that had been frozen for up to four days. [ 5 ]  The third method of cloning involves embryo splitting, the process of taking the  blastomeres  from a very early animal embryo and separating them before they become  differentiated  in order to create two or more separate organisms. When using embryo splitting, cloning must occur before the birth of the animal, and clones grow up at the same time (in a similar fashion to  monozygotic twins ). [ 3 ]


The  US Food and Drug Administration  has concluded that "Food from cattle, swine, and goat clones is as safe to eat as food from any other cattle, swine, or goat." [ 1 ]   It has also been noted that the main use of agricultural clones is to produce breeding stock, not food. Clones allow farmers to upgrade the overall quality of their herds by producing more copies of the best animals in the herd. These animals are then used for conventional breeding, and the sexually reproduced offspring become the food producing animals. The goals of cloning listed by the FDA include "disease resistance ... suitability to climate ... quality body type .. fertility ... and market preference (leanness, tenderness, color, size of various cuts, etc.)" [ 1 ]  Milk productivity is another desirable trait that cloning is used for, including in the case of cloned " supercows ". [ 6 ]


Organs from cloned pigs have been transplanted into human patients. [ 7 ] [ better source needed ]  (See  Xenotransplantation )


Cancer-sniffing dogs have also been cloned. A  review  concluded that "qualified  elite  working dogs can be produced by cloning a working dog that exhibits both an appropriate temperament and good health." [ 8 ]


Cloning of super  sniffer dogs  for airports was reported in 2011, four years after the dog that served as their genetic donor retired. [ 9 ]  Cloning of a successful rescue dog was reported in 2009 [ 10 ]  and of a police dog in 2019. [ 11 ]


The only extinct animal to be cloned as of 2022 is a  Pyrenean ibex , born on July 30, 2003, in  Spain , which died minutes later due to physical defects in the lungs. [ 12 ] [ 13 ]


Some animals have been cloned to add genetic diversity to endangered species with small remaining populations, thereby avoiding  inbreeding depression .  Centers performing this include  ViaGen , aided by the  San Diego Frozen Zoo , and  Revive & Restore . [ 14 ]   This is also referred to as "conservation cloning". [ 15 ] [ 16 ]   Two examples are the  black-footed ferret  and  Przewalski's horse . [ 14 ]


In 2022, the world's first cloned Arctic wolf "Maya" was born in  Beijing  by  Sinogene . Although  Arctic wolves  are no longer listed by the  IUCN Red List  as an endangered species, the technique can be used to help other animals at risk of extinction, such as  Mexican gray wolves  and  red wolves . The team of Sinogene plans to restore lost species or boost numbers in endangered animal populations. [ 17 ]


In a recent study using  sturgeons , scientists have made improvements to a technique known as somatic nuclear cell transfer, with the ultimate goal being to save endangered species. Sturgeons are endangered due to the high levels of  poaching , increased destruction to habitats, water pollution, and overfishing. The somatic nuclear cell transfer technique is a well-known cloning method that has been used for years but focuses on species that are thriving rather than endangered or extinct species. This technique usually uses a single somatic donor cell with a single manipulation and inserts it into a recipient egg of the species of interest. It has recently been found that the position by which that somatic cell is located inside the recipient is very important in order to successfully clone a species. By making adjustments to the original method of using a single somatic cell and instead use multiple somatic donor cells to insert into the recipient egg, the likeliness of the somatic donor cells being in the crucial position on the egg will increase tremendously. This increase will then result in higher success rates with cloning. There is ongoing research using this improved method, but from the data collected thus far, it seems to be a reasonable method to continue and soon be able to help stop species like the sturgeons from becoming endangered and possibly stop extinction from occurring. [ 18 ]


Cloning long-extinct animals using current methods is impossible because DNA begins to  denature  after death, meaning the entire genome of an extinct species is not available to be reproduced. However, new studies using  genome editing  have suggested it may be possible to "bring back" traits of extinct species by incorporating genes from the extinct species into the genome of a closely related living organism. Currently, George Church's lab at  Harvard University 's Wyss Institute is conducting research into genetically modifying  Asian elephants  to express genes from the extinct  woolly mammoth . [ 19 ]  Their goals in doing this are to expand the habitat available to Asian elephants and reestablish the ecological interactions woolly mammoths played a role in prior to their extinction.


ViaGen  began by offering cloning to the livestock and equine industry in 2003, [ 20 ]  and later as  ViaGen Pets  included cloning of cats and dogs in 2016. [ 21 ]   ViaGen's  subsidiary, start licensing, owns a cloning patent which is licensed to their only competitor as of 2018, who also offers animal cloning services. [ 22 ]  (Viagen is a subsidiary of  Precigen . [ 23 ] )


The first commercially cloned pet was a cat named  Little Nicky , produced in 2004 by  Genetic Savings & Clone  for a north  Texas  woman for the fee of  US$ 50,000. [ 24 ]   On May 21, 2008, BioArts International [ 25 ]  announced a limited commercial dog cloning service (through a program it called Best Friends Again) in partnership with a Korean company named  Sooam Biotech .  This program came after the announcement of the successful cloning of a family dog named Missy, an achievement widely publicized in the  Missyplicity  Project. In September 2009, BioArts announced the end of its dog cloning service. [ 26 ]  In July 2008, the  Seoul National University  (co-parents of  Snuppy , reputedly the world's first cloned dog in 2005) created five clones of a dog named Booger for its Californian owner. The woman paid $50,000 for this service. [ 27 ]


Sooam Biotech continued developing proprietary techniques for cloning dogs [ 28 ]  based on a licence from  ViaGen's  subsidiary, stART Licensing (which owned the original patent for the process of animal cloning [ 29 ] ). (Although the animal itself is not patentable, the process is protected by a patent). [ 30 ]  Sooam created cloned puppies for owners whose dogs had died, charging $100,000 per clone. [ 31 ] [ 32 ]  Sooam Biotech was reported to have cloned approximately 700 dogs by 2015 [ 31 ]  and to be producing 500 cloned embryos of various breeds a day in 2016. [ 33 ]  In 2015, the longest period after which Sooam Biotech could clone a puppy was 12 days from the death of the original pet dog. [ 34 ]
Sinogene Biotechnology created the first Chinese clone dog in 2017 before commercializing the cloning service and joining in the pet cloning market. [ 35 ]  In 2019, Sinogene successfully created the first Chinese cloned cat. [ 36 ]  In June 2022, "Zhuang Zhuang" was cloned by the Beijing laboratory Sinogene. He is the first from the "warmblood" group of breeds to be born in  China  and to be officially approved by the China Horse Industry Association. [ 37 ]


The mortality rate for cloned animals is higher than for those born of natural processes. This includes a discrepancy pre-birth, during birth, and after birth in survival rates and quality of life, leading to ethical concerns. [ 38 ]  Many of these discrepancies are thought to come from  maternal mRNA  already present in the oocyte prior to the transfer of genetic material as well as from  DNA methylation , both of which contribute to the development of the animal in the womb of the surrogate. [ 3 ]  Some common issues seen with cloned animals are shortened  telomeres , the repetitive end sequences of DNA whose decreasing length over the lifespan of an organism have been associated with aging; [ 39 ]  large offspring syndrome, the abnormal size of cloned individuals due to  epigenetic  (gene expression) changes; and methylation patterns of genetic material that are so abnormal compared to standard embryos of the species being cloned as to be incompatible with life. [ 3 ]


While pet cloning is sometimes advertised as a prospective method for re-gaining a deceased companionship animal, [ 40 ]  pet cloning does not result in animals that are exactly like the previous pet (in looks or personality). [ 41 ]  Although the animal in question is cloned, there are still  phenotypical  differences that may affect its appearance or health. This issue was brought to light in the cloning of a cat named Rainbow. Rainbow's clone, later named CC, was genetically identical to Rainbow, yet CC's coloring patterns were not the same due to the development of the kitten inside the womb as well as random genetic disparities in the clone such as variable  X-chromosome inactivation . [ 42 ]


Despite its controversies, the study of pet cloning holds the potential to contribute to  scientific ,  veterinary , and  medical  knowledge, and it is a potential resource in efforts to preserve endangered cousins of the cat and dog. [ 39 ]


In 2005,  California Assembly  Member  Lloyd Levine  introduced a bill to ban the sale or transfer of pet clones in  California . [ 43 ]  That bill was voted down. [ 44 ]


A plant  cutting  is a piece of a plant that is used in  horticulture  for  vegetative  (asexual)  propagation . A piece of the  stem  or root of the source plant is placed in a suitable medium such as moist  soil . If the conditions are suitable, the plant piece will begin to grow as a new plant independent of the  parent , a process known as  striking . A stem cutting produces new  roots , and a root cutting produces new stems. Some plants can be grown from leaf pieces, called leaf cuttings, which produce both stems and roots. The scions used in  grafting  are also called cuttings. [ 1 ]


Propagating plants from cuttings is an ancient form of  cloning . [ 2 ] [ 3 ]  There are several advantages of cuttings, mainly that the produced offspring are practically clones of their parent plants. If a plant has favorable traits, it can continue to pass down its advantageous  genetic information  to its offspring. This is especially economically advantageous as it allows commercial growers to clone a certain plant to ensure consistency throughout their crops. [ 4 ]


Cuttings are used as a method of  asexual reproduction  in succulent horticulture, commonly referred to as  vegetative reproduction . A cutting can also be referred to as a  propagule . Succulents have evolved with the ability to use adventitious root formation in reproduction to increase fitness in stressful environments.  Succulents  grow in shallow soils, rocky soils, and desert soils. [ 5 ]  Seedlings from sexual reproduction have a low survival rate; however, plantlets from the excised stem cuttings and leaf cuttings, broken off in the natural environment, are more successful. [ 5 ]


Cuttings have both water and carbon stored and available, which are resources needed for plant establishment. The detached part of the plant remains physiologically active, allowing mitotic activity and new root structures to form for water and nutrient uptake. [ 5 ]  Asexual reproduction of plants is also evolutionarily advantageous as it allows plantlets to be better suited to their environment through retention of  epigenetic  memory, heritable patterns of phenotypic differences that are not due to changes in DNA but rather  histone  modification and  DNA methylation . [ 6 ]  Epigenetic memory is heritable through mitosis, and thus advantageous stress response priming is retained in plantlets from excised stem. [ 6 ]


Adventitious root formation refers to roots that form from any structure of a plant that is not a root; these roots can form as part of normal development or due to a stress response. [ 7 ]  Adventitious root formation from the excised stem cutting is a wound response.


At a molecular level when a cutting is first excised at the stem there is an immediate increase in  jasmonic acid , known to be necessary for adventitious root formation. [ 8 ]  When the cutting is excised from the original root system the root inhibiting hormones,  cytokinin  and  strigolactone , which are made in the root and transported to the stem, decrease in concentration. [ 7 ]  Polyphenol degradation decreases, increasing auxin concentration. The increased  auxin  concentration increases nitric oxide concentration which initiates root formation through a  MAPK signal cascade  and a  cGMP-dependent pathway  that both regulate mitotic division and are both necessary for the initiation of adventitious root formation. [ 9 ]  The root  primordia  form from  cambial  cells in the stem. [ 7 ]  In propagation of detached succulent leaves and leaf cuttings, the root primordia typically emerges from the basal callous tissue after the leaf primordia emerges. [ 5 ]


It was known as early as 1935 that when  indolyl-3-acetic acid  (IAA), also known as  auxin , is applied to the stem of root cuttings, there is an increase in the average number of adventitious roots compared to cuttings that are not treated. Researchers also applied this compound to stems without leaves that normally would not have any root formation and found that auxin induced root formation, thus determining auxin is necessary for root formation. [ 10 ]  Identification of this hormone has been important to industries that rely on vegetative propagation, [ 7 ]  as it is sometimes applied to fresh cuttings to stimulate root growth.


Some plants form roots much more easily than others. Stem cuttings from woody plants are treated differently, depending on the maturity of the wood:


Most plant cuttings are stem pieces, and have no root system of their own,  and are therefore likely to die from  dehydration  if the proper conditions are not met. They require a moist medium, which, however, cannot be too wet lest the cutting rot. A number of media are used in this process, including but not limited to soil,  perlite ,  vermiculite ,  coir ,  rock wool ,  expanded clay pellets , and even water given the right conditions. Most  succulent  cuttings can be left in open air until the cut surface dries, which may improve root formation when the cutting is later planted.


In  temperate countries , stem cuttings may be taken of soft (green or semi-ripe) wood and hard wood, which have specific differences in practice. Certain conditions lead to more favorable outcomes for cuttings; timing, size, location on the plant, and amount of foliage are all important. Stem cuttings of young wood should be taken in spring from the upper branches, while cuttings of hardened wood should be taken in winter from the lower branches. Common bounds on the length of stem cuttings are between 5–15 centimetres (2.0–5.9 in) for soft wood and between 20–25 centimetres (7.9–9.8 in) for hard wood. Soft wood cuttings do best when about two thirds of the foliage are removed, [ 13 ]  while hard wood stem cuttings need complete foliage removal. The cut needs to be done either immediately below a node, [ 14 ]  or up to 1/2 inch below a node. [ 15 ]


Besides placing the cuttings directly into soil, it is also possible to root cuttings in water. The water needs to be replaced often, to prevent bacteria buildup and the possibility of  root rot . It also requires enough oxygen in the water for the same reason. A moist atmosphere (use of plastic sheeting) is hence not needed with this technique. [ 16 ] [ 17 ]


The environment for softwood and semi-hardwood cuttings is generally kept  humid —often attained by placing the cuttings under a plastic sheet or in another confined space where the air can be kept moist—and in partial shade to prevent the cutting from drying out. Cuttings in the medium are typically watered with a fine  mist  to avoid disturbing the plants. Following the initial watering, the aim is to keep the soil moist but not wet or waterlogged; the medium is allowed to almost dry out before misting again. [ 13 ]


A  rooting hormone  may be administered to "encourage" growth and can increase the success rate of plant growth. [ 18 ]  Though not essential, several compounds may be used to promote the formation of roots through the signaling activity of  plant hormone   auxins . Among the commonly used chemicals is  indole-3-butyric acid  (IBA) used as a powder, liquid solution, or gel. This compound is applied either to the cut tip of the cutting or as a foliar spray. Rooting hormone can be manufactured naturally, such as soaking the yellow-tipped shoots of a weeping willow tree in water, or by preparing a  tea  from the bark of a willow tree. Shoots or bark do better when soaked for 24 hours prior to using. [ 19 ]  The extract obtained from the crushing of leaves and bulbs of  coco-grass  ( Cyperus rotundus ) is used as an excellent rooting of cuttings and seedlings of various plant species. [ 20 ] [ 21 ]   Honey , though it does not contain any plant hormones, can also aid in rooting success through its natural  antiseptic  and  antifungal  properties. [ 22 ] [ 23 ]   Cinnamon [ citation needed ]  or an  Aspirin  tablet in water [ 24 ]  can also aid the rooting process. [ 25 ]


Many vegetative parts of a plant can be used. The most common methods are:


Although some species, such as  willow ,  blackberry  and  pelargoniums  can be grown simply by placing a cutting into moist ground, the majority of species require more attention. Most species require humid, warm, partially shaded conditions to strike, thus requiring the approach above to be followed. Particularly difficult species may need cool air above and warm soil. In addition, with many more difficult cuttings, one should use the type of cutting that has the most chance of success with that particular plant species. [ 28 ]


There are ways of improving the growth of stem cutting propagations. Intensifying light allows cuttings to root and sprout faster, though the heat thus generated could cause the propagation material distress. [ 29 ]   Azalea  cuttings can be mildly heated in water to disinfect it from the fungus pathogen  Rhizoctonia , and this could potentially be used for other plants. [ 30 ]


Depending on the type of cutting (i.e. tree, shrub, succulent, cacti) different  potting soil  mixes can be used. Many commercial companies sell medium specifically for growing cuttings.


Although several options can be used here, usually plastic is used to cover the softwood and semi-hardwood cuttings. The soil below the trays (to increase air moisture) and the soil in the trays themselves is kept moist but not waterlogged (=completely saturated). The trays the cuttings sit in are best placed on stones to prevent capillary action (as this can keep the soil inside the trays too wet). Soil in the trays should be kept at 85 to 95% saturation. [ 31 ]  Automated (overhead) misting systems, boom systems or fog systems [ 32 ]  can be used in greenhouses.


A typical misting frequency during sticking and callusing includes misting for 5–8 seconds every 5–10 minutes over a 24-hour period. After 3 to 4 days, misting is reduced to 3–5 seconds every 10–20 minutes during the day and less frequently at night. When roots become visible (stage 3) misting can be reduced, and by stage 4 (toning), little to no misting should be done (by day 10 to 14 for most species [ 31 ] ). [ 32 ]  When using plastic tents, far less misting is needed (once or twice a day). [ 33 ]  The greenhouse or cold frame should be ventilated once in a while [ 34 ]  to prevent formation of  molds  (manually).


Air temperature for softwood and semi-hardwood cuttings is optimal at around 70 °F [ 35 ] [ 36 ] [ 37 ] [ 38 ]  (21.1 °C) but temperatures as low as 55 °F (12.7 °C) are acceptable. Heating the air above 75 °F (23.8 °C) stimulates the growth of pathogens. [ 35 ]  Ventilating (manually or through automatic window openers) the greenhouse or cold frame can lower the air temperature. Automated thermostat systems can also be used in greenhouses to keep the heat at a specific temperature. Bottom heating (soil) tends to be ideal for root initiation since growing media temperature is best maintained at 20-22 °C. [ 31 ]


Whereas cuttings need to be kept warm and some amount of light needs to be provided, it needs to be kept out of direct sunlight. [ 39 ]  Some ways to accomplish this include using white wash, semi-white plastic, retractible shade curtains (which can be deployed if the sun temporarily pierces through), ...
Optimum light levels are around 120 to 200 μmol/m 2 s at the first stage (sticking [ 32 ] ). Once callus has been formed (stage 2: callusing [ 32 ] ) and roots start to form and take up water (stage 3: root development phase), light intensity levels can be gradually increased (to 200 to 800 μmol/m 2 s). [ 40 ] [ 41 ]  Most propagators find that 5 to 10 moles per day (i.e. observed using a Daily Light Integral sensor) will result in a consistent rooting and growth [ 31 ]


Plants which can be propagated from stem, leaf and/or tip cuttings include: [ 42 ] [ failed verification  –  see discussion ]


To note, some plants listed above, such as Coleus, Maranta, Nerium and Golden Pothos, among others, may require water for them to root before they can be transplanted to soil.


The poet  Theodore Roethke  wrote about plant cuttings and root growth behavior in his poems "Cuttings" and "Cuttings (Later)"  found in his book  The Lost Son: And Other Poems. [ 43 ]


De-extinction  (also known as  resurrection biology , or  species revivalism ) is the process of generating an  organism  that either resembles or is an  extinct species . [ 1 ]  There are several ways to carry out the process of de-extinction.  Cloning  is the most widely proposed method, although genome editing and  selective breeding  have also been considered. Similar techniques have been applied to certain  endangered species , in hopes to boost their  genetic diversity . The only method of the three that would provide an animal with the same genetic identity is cloning. [ 2 ]  There are benefits and drawbacks to the process of de-extinction ranging from technological advancements to ethical issues.


Cloning  is a commonly suggested method for the potential restoration of an extinct species. It can be done by extracting the nucleus from a preserved cell from the extinct species and swapping it into an egg, without a nucleus, of that species' nearest living relative. [ 3 ]  The egg can then be inserted into a host from the extinct species' nearest living relative. This method can only be used when a preserved cell is available, meaning it would be most feasible for recently extinct species. [ 4 ]  Cloning has been used by scientists since the 1950s. [ 5 ]  One of the most well known clones is  Dolly the sheep . Dolly was born in the mid 1990s and lived normally until the abrupt midlife onset of health complications resembling premature aging, that led to her death. [ 5 ]  Other known cloned animal species include domestic cats, dogs, pigs, and horses. [ 5 ]


Genome editing  has been rapidly advancing with the help of the CRISPR/Cas systems, particularly CRISPR/Cas9. The CRISPR/Cas9 system was originally discovered as part of the bacterial immune system. [ 6 ]  Viral DNA that was injected into the bacterium became incorporated into the bacterial chromosome at specific regions. These regions are called clustered regularly interspaced short palindromic repeats, otherwise known as CRISPR. Since the viral DNA is within the chromosome, it gets transcribed into RNA. Once this occurs, the Cas9 binds to the RNA. Cas9 can recognize the foreign insert and cleaves it. [ 6 ]  This discovery was very crucial because now the Cas protein can be viewed as a scissor in the genome editing process.


By using cells from a closely related species to the extinct species, genome editing can play a role in the de-extinction process. Germ cells may be edited directly, so that the egg and sperm produced by the extant parent species will produce offspring of the extinct species, or somatic cells may be edited and transferred via somatic cell nuclear transfer. The result is an animal which is not completely the extinct species, but rather a hybrid of the extinct species and the closely related, non-extinct species. Because it is possible to sequence and assemble the genome of extinct organisms from highly degraded tissues, this technique enables scientists to pursue de-extinction in a wider array of species, including those for which no well-preserved remains exist. [ 3 ]  However, the more degraded and old the tissue from the extinct species is, the more fragmented the resulting DNA will be, making genome assembly more challenging.


Back breeding is a form of selective breeding. As opposed to breeding animals for a trait to advance the species in selective breeding, back breeding involves breeding animals for an ancestral characteristic that may not be seen throughout the species as frequently. [ 7 ]  This method can recreate the traits of an extinct species, but the genome will differ from the original species. [ 4 ]  Back breeding, however, is contingent on the ancestral trait of the species still being in the population in any frequency. [ 7 ]  Back breeding is also a form of artificial selection by the deliberate selective breeding of domestic animals, in an attempt to achieve an animal breed with a phenotype that resembles a wild type ancestor, usually one that has gone extinct.


A natural process of de-extinction is iterative evolution. This occurs when a species becomes extinct, but then after some time a different species evolves into an almost identical creature. For example, the Aldabra rail was a flightless bird that lived on the island of  Aldabra . It had evolved some time in the past from the flighted  white-throated rail , but became extinct about 136,000 years ago due to an unknown event that caused sea levels to rise. About 100,000 years ago, sea levels dropped and the island reappeared, with no fauna. The white-throated rail recolonized the island, but soon evolved into a flightless species physically identical to the extinct species. [ 8 ] [ 9 ]


Not all extinct plants have herbarium specimens that contain seeds. Of those that do, there is ongoing discussion on how to coax barely alive embryos back to life. [ 10 ]  See  Judean date palm  and  tsori .


In-vitro fertilisation and artificial insemination are  assisted reproduction technology  commonly used to treat  infertility  in humans. However, it has usage as a viable option for de-extinction in cases of  functional extinction  where all remaining individuals are of the same sex, incapable of naturally reproducing, or suffer from low genetic diversity such as the northern white rhinoceros, Yangtze giant softshell turtle,  Hyophorbe amaricaulis , baiji, and vaquita.  [ 11 ]  For example, viable embryos are created from preserved sperm from deceased males and  ova  from living females are implemented into a surrogate species. [ 12 ]


The technologies being developed for de-extinction could lead to large advances in various fields:


Prioritising de-extinction could lead to the improvement of current conservation strategies. Conservation measures would initially be necessary in order to reintroduce a species into the ecosystem, until the revived population can sustain itself in the wild. [ 16 ]  Reintroduction of an extinct species could also help improve ecosystems that had been destroyed by human development. It may also be argued that reviving species driven to extinction by humans is an ethical obligation. [ 17 ]


The reintroduction of extinct species could have a negative impact on extant species and their ecosystem. The extinct species' ecological niche may have been filled in its former habitat, making it an invasive species. This could lead to the extinction of other species due to competition for food or other  competitive exclusion . It could lead to the extinction of prey species if they have more predators in an environment that had few predators before the reintroduction of an extinct species. [ 17 ]  If a species has been extinct for a long period of time the environment they are introduced to could be wildly different from the one that they can survive in. The changes in the environment due to human development could mean that the species may not survive if reintroduced into that ecosystem. [ 13 ]  A species could also become extinct again after de-extinction if the reasons for its extinction are still a threat. The woolly mammoth might be hunted by poachers just like  elephants  for their  ivory  and could go extinct again if this were to happen. Or, if a species is reintroduced into an environment with disease for which it has no immunity, the reintroduced species could be wiped out by a disease that current species can survive.


De-extinction is a very expensive process. Bringing back one species can cost millions of dollars. The money for de-extinction would most likely come from current conservation efforts. These efforts could be weakened if funding is taken from conservation and put into de-extinction. This would mean that  critically endangered  species would start to go extinct faster because there are no longer resources that are needed to maintain their populations. [ 18 ]  Also, since cloning techniques cannot perfectly replicate a species as it existed in the wild, the reintroduction of the species may not bring about positive environmental benefits. They may not have the same role in the food chain that they did before and therefore cannot restore damaged ecosystems. [ 19 ]


The existence of preserved soft tissue remains and  DNA  from  woolly mammoths  ( Mammuthus primigenius ) has led to the idea that the species could be recreated by scientific means. Two methods have been proposed to achieve this:


The first would be to use the cloning process; [ 20 ]  however, even the most intact mammoth samples have had little usable DNA because of their conditions of preservation. There is not enough DNA intact to guide the production of an embryo. [ 21 ]  The second method would involve  artificially inseminating  an elephant egg cell with preserved sperm of the mammoth. The resulting offspring would be a  hybrid  of the mammoth and its closest living relative the  Asian elephant . After several generations of cross-breeding these hybrids, an almost pure woolly mammoth could be produced. However, sperm cells of modern mammals are typically potent for up to 15 years after deep-freezing, which could hinder this method. [ 22 ]   Whether the hybrid embryo would be carried through the two-year gestation is unknown; in one case, an Asian elephant and an African elephant produced a live calf named  Motty , but it died of defects at less than two weeks old. [ 23 ]


In 2008, a Japanese team found usable DNA in the brains of mice that had been frozen for 16 years. They hope to use similar methods to find usable mammoth DNA. [ 24 ]  In 2011, Japanese scientists announced plans to clone mammoths within six years. [ 25 ]


In March 2014, the Russian Association of Medical Anthropologists reported that blood recovered from a frozen mammoth carcass in 2013 would now provide a good opportunity for cloning the woolly mammoth. [ 22 ]  Another way to create a living woolly mammoth would be to migrate genes from the mammoth genome into the genes of its closest living relative, the Asian elephant, to create hybridized animals with the notable adaptations that it had for living in a much colder environment than modern day elephants. [ 26 ]   This is currently being done by a team led by Harvard geneticist  George Church . [ 27 ]  The team has made changes in the elephant genome with the genes that gave the woolly mammoth its cold-resistant blood, longer hair, and an extra layer of fat. [ 27 ]  According to geneticist Hendrik Poinar, a revived woolly mammoth or mammoth-elephant hybrid may find suitable habitat in the tundra and taiga forest ecozones. [ 28 ]


George Church has hypothesized the positive effects of bringing back the extinct woolly mammoth would have on the environment, such as the potential for reversing some of the damage caused by  global warming . [ 29 ]  He and his fellow researchers predict that mammoths would eat the dead grass allowing the sun to reach the spring grass; their weight would allow them to break through dense, insulating snow in order to let cold air reach the soil; and their characteristic of felling trees would increase the absorption of sunlight. [ 29 ]  In an editorial condemning de-extinction,  Scientific American  pointed out that the technologies involved could have secondary applications, specifically to help species on the verge of extinction regain their  genetic diversity . [ 30 ]


The  Pyrenean ibex  ( Capra pyrenaica pyrenaica ) was a subspecies of  Iberian ibex  that lived on the Iberian Peninsula. While it was abundant through  medieval times , over-hunting in the 19th and 20th centuries led to its demise. In 1999, only a single female named Celia was left alive in  Ordesa National Park . Scientists captured her, took a tissue sample from her ear, collared her, then released her back into the wild, where she lived until she was found dead in 2000, having been crushed by a fallen tree.


In 2003, scientists used the tissue sample to attempt to clone Celia and resurrect the extinct subspecies. Despite having successfully transferred nuclei from her cells into  domestic goat  egg cells and impregnating 208 female goats, only one came to term. The baby ibex that was born had a lung defect, and lived for only seven minutes before suffocating from being incapable of breathing oxygen. Nevertheless, her birth was seen as a triumph and is considered the first de-extinction. [ 31 ]  In late 2013, scientists announced that they would again attempt to resurrect the Pyrenean ibex. [ 32 ] [ 33 ]


A problem to be faced, in addition to the many challenges of reproduction of a mammal by cloning, is that only females can be produced by cloning the female individual Celia, and no males exist for those females to reproduce with. This could potentially be addressed by breeding female clones with the closely related  Southeastern Spanish ibex , and gradually creating a hybrid animal that will eventually bear more resemblance to the Pyrenean ibex than the Southeastern Spanish ibex. [ 32 ]


The  aurochs  ( Bos primigenius ) was widespread across Eurasia, North Africa, and the Indian subcontinent during the  Pleistocene , but only the European aurochs ( B. p. primigenius ) survived into historical times. [ 34 ]  This species is heavily featured in European cave paintings, such as  Lascaux  and  Chauvet  cave in France, [ 35 ]  and was still widespread during the  Roman era . Following the fall of the  Roman Empire , overhunting of the aurochs by nobility caused its population to dwindle to a single population in the  Jaktorów  forest in Poland, where the last wild one died in 1627. [ 36 ]


However, because the aurochs is ancestral to most modern  cattle  breeds, it is possible for it to be brought back through selective or back breeding. The first attempt at this was by  Heinz  and  Lutz Heck  using modern cattle breeds, which resulted in the creation of  Heck cattle . This breed has been introduced to nature preserves across Europe; however, it differs strongly from the aurochs in physical characteristics, and some modern attempts claim to try to create an animal that is nearly identical to the aurochs in morphology, behavior, and even genetics. [ 37 ]  There are several projects that aim to create a cattle breed similar to the aurochs through selectively breeding primitive cattle breeds over a course of twenty years to create a self-sufficient bovine grazer in herds of at least 150 animals in rewilded nature areas across Europe, for example the  Tauros Programme  and the separate  Taurus Project . [ 38 ]  This organization is partnered with the organization  Rewilding Europe  to help revert some European natural ecosystems to their prehistoric form. [ 39 ]


A competing project to recreate the aurochs is the  Uruz Project  by the  True Nature Foundation , which aims to recreate the aurochs by a more efficient breeding strategy using  genome editing , in order to decrease the number of generations of breeding needed and the ability to quickly eliminate undesired traits from the population of aurochs-like cattle. [ 40 ]  It is hoped that aurochs-like cattle will reinvigorate European nature by restoring its ecological role as a keystone species, and bring back biodiversity that disappeared following the decline of European  megafauna , as well as helping to bring new economic opportunities related to European wildlife viewing. [ 41 ]


The  quagga  ( Equus quagga quagga ) is a subspecies of the  plains zebra  that was distinct in that it was striped on its face and upper torso, but its rear abdomen was a solid brown. It was native to  South Africa , but was wiped out in the wild due to overhunting for sport, and the last individual died in 1883 in the Amsterdam Zoo. [ 42 ]  However, since it is technically the same species as the surviving plains zebra, it has been argued that the quagga could be revived through artificial selection. The  Quagga Project  aims to breed a similar form of zebra by selective breeding of plains zebras. [ 43 ]  This process is also known as back breeding. It also aims to release these animals onto the western Cape once an animal that fully resembles the quagga is achieved, which could have the benefit of eradicating  introduced species  of  trees  such as the  Brazilian pepper tree ,  Tipuana tipu ,  Acacia saligna ,  bugweed ,  camphor tree ,  stone pine ,  cluster pine ,  weeping willow  and  Acacia mearnsii . [ 44 ]


The  thylacine  ( Thylacinus cynocephalus ), commonly known as the Tasmanian tiger, was native to the  Australian mainland ,  Tasmania  and  New Guinea . It is believed to have become  extinct  in the 20th century. The thylacine had become extremely rare or extinct on the Australian mainland before  British settlement  of the continent. The last known thylacine died at the  Hobart Zoo , on September 7, 1936. He is believed to have died as the result of neglect—locked out of his sheltered sleeping quarters, he was exposed to a rare occurrence of extreme Tasmanian weather:  extreme heat  during the day and freezing temperatures at night. [ 45 ]  Official protection of the species by the Tasmanian government was introduced on July 10, 1936, roughly 59 days before the last known specimen died in captivity. [ 46 ]


In December 2017, it was announced in the journal  Nature Ecology and Evolution  that the full nuclear genome of the thylacine had been successfully sequenced, marking the completion of the critical first step toward de-extinction that began in 2008, with the extraction of the DNA samples from the preserved pouch specimen. [ 47 ]  The thylacine genome was reconstructed by using the genome editing method. The  Tasmanian devil  was used as a reference for the assembly of the full nuclear genome. [ 48 ]  Andrew J. Pask from the  University of Melbourne  has stated that the next step toward de-extinction will be to create a functional genome, which will require extensive research and development, estimating that a full attempt to resurrect the species may be possible as early as 2027. [ 47 ]


In August 2022, the University of Melbourne and  Colossal Biosciences  announced a partnership to accelerate de-extinction of the thylacine via genetic modification of one of its closest living relatives, the  fat-tailed dunnart . [ 49 ]  In 2024, a 99.9% complete genome of the thylacine was created from a well-preserved skull that is estimated to be 110 years old. [ 50 ]


The  passenger pigeon  ( Ectopistes migratorius ) numbered in the billions before being wiped out due to unsustainable commercial hunting and habitat loss during the early 20th century. The non-profit  Revive & Restore  obtained DNA from the passenger pigeon from museum specimens and skins; however, this DNA is degraded because it is so old. For this reason, simple cloning would not be an effective way to perform de-extinction for this species because parts of the genome would be missing. Instead, Revive & Restore focuses on identifying mutations in the DNA that would cause a phenotypic difference between the extinct passenger pigeon and its closest living relative, the  band-tailed pigeon . In doing this, they can determine how to modify the DNA of the band-tailed pigeon to change the traits to mimic the traits of the passenger pigeon. In this sense, the de-extinct passenger pigeon would not be genetically identical to the extinct passenger pigeon, but it would have the same traits. In 2015, the de-extinct passenger pigeon hybrid was forecast ready for captive breeding by 2025 and released into the wild by 2030. [ 51 ]


The  bush moa , also known as the little bush moa or lesser moa  (Anomalopteryx didiformis)  is a slender species of  moa  slightly larger than a  turkey  that went extinct abruptly, around 500–600 years ago following the arrival and proliferation of the  Māori people  in  New Zealand , as well as the introduction of  Polynesian dogs . [ 52 ]  Scientists at  Harvard University  assembled the first nearly complete genome of the species from toe bones, thus bringing the species a step closer to de-extinction. [ 53 ]  The New Zealand politician,  Trevor Mallard  has also previously suggested bringing back a medium-sized species of moa. [ 54 ]  The proxy of the species will likely be the emu. [ 55 ]


The  Maclear's rat  ( Rattus macleari ), also known as the Christmas Island rat, was a large rat endemic to Christmas Island in the Indian Ocean. It is believed Maclear's rat might have been responsible for keeping the population of  Christmas Island red crab  in check. It is thought that the accidental introduction of black rats by the  Challenger expedition  infected the Maclear's rats with a disease (possibly a trypanosome), [ 56 ]  which resulted in the species' decline. [ 57 ]  The last recorded sighting was in 1903. [ 58 ]  In March 2022, researchers discovered the Maclear's rat shared about 95% of its genes with the living  brown rat , thus sparking hopes in bringing the species back to life. Although scientists were mostly successful in using CRISPR technology to edit the DNA of the living species to match that of the extinct one, a few key genes were missing, which would mean resurrected rats would not be genetically pure replicas. [ 59 ]


The  dodo  ( Raphus cucullatus ) was a flightless bird endemic to the island of Mauritius in the Indian Ocean. Due to various factors such as the inability to  feel fear  caused by isolation from significant predators, predation from humans and introduced invasive species such as pigs, dogs, cats, rats, and  crab-eating macaques , competition for food with invasive species, habitat loss, and the birds naturally slow reproduction, the species' numbers declined rapidly. [ 60 ]  The last widely accepted recorded sighting was in 1662. Since then, the bird has become a symbol for extinction and is often cited as the primary example of  man-made extinction . [ 61 ]  In January 2023,  Colossal Biosciences  announced their project to revive the dodo alongside their previously announced projects for reviving the woolly mammoth and thylacine in hopes of restoring biodiversity to Mauritius and changing the dodo's status as a symbol of extinction to de-extinction. [ 62 ]   [ 63 ]


The  Steller's sea cow  was a  sirenian  endemic to  Bering Sea  between Russia and the United States but had a much larger range during the  Pleistocene . First described by  Georg Wilhelm Steller  in 1741, it was hunted to extinction 27 years later due to its  buoyancy  making it an easy target for humans hunting it for its meat and fur in addition to an already low population caused by climate change. In 2021, the nuclear genome of the species was sequenced. [ 64 ]  In late 2022, a group of Russian scientists funded by Sergei Bachin began their project to revive and reintroduce the giant sirenian to its former range in the 18th century to restore its kelp forest ecosystem. Arctic Sirenia plans to revive the species through genome editing of the  dugong , but they need an  artificial womb  to conceive a live animal due to lack of an adequate surrogate species. [ 65 ]   Ben Lamm  of Colossal Biosciences has also expressed desire to revive the species once his company develops an artificial womb. [ 66 ]


The  northern white rhinoceros  or northern white rhino  (Ceratotherium simum cottoni)  is a subspecies of the  white rhinoceros  endemic to East and Central Africa south of the  Sahara . Due to widespread and uncontrollable poaching and civil warfare in their former range, the subspecies' numbers dropped quickly over the course of the late 1900s and early 2000s. [ 67 ] [ 68 ]  Unlike the majority of the potential candidates for de-extinction, the northern white rhinoceros is not extinct, but  functionally extinct  and is believed to be extinct in the wild with only two known female members left, Najin and Fatu who reside on the  Ol Pejeta Conservancy  in Kenya. [ 69 ]  The BioRescue Team in collaboration with  Colossal Biosciences  plan to implement 30 northern white rhinoceros embryos made from egg cells collected from Najin and Fatu and preserved sperm from dead male individuals into female  southern white rhinoceros  by the end of 2024. [ 12 ] [ 70 ]


The  ivory-billed woodpecker   (Campephilus principalis)  is the largest woodpecker endemic to the United States with a  subspecies  in Cuba. The species numbers have declined since the late 1800s due to  logging  and hunting. [ 71 ]  Similar to the northern white rhinoceros, the ivory-billed woodpecker is not completely extinct, but  functionally extinct  with occasional sightings that suggest that 50 or less individuals are left. [ 72 ]  In October 2024, Colossal Biosciences announced their non-profit Colossal Foundation, a foundation dedicated to conservation of extant species with their first projects being the  Sumatran rhinoceros ,  vaquita ,  red wolf ,  pink pigeon ,  northern quoll , and ivory-billed woodpecker. Colossal plans to revive or rediscover the species through genome editing of its closest living relatives and using  drones  and  AI  to identify any potential remaining individuals in the wild. [ 73 ] [ 74 ]


The  heath hen   (Tympanuchus cupido cupido)  was a subspecies of  greater prairie chicken  endemic to the  heathland barrens  of coastal North America. It is even speculated that the  pilgrims ' first  Thanksgiving  featured this bird as the main course instead of  wild turkey . [ 75 ]  Due to overhunting caused by its perceived abundancy, the population  became extinct in mainland North America  by 1870, leaving a population of 300 individuals left on  Martha's Vineyard . Despite conservation efforts, the subspecies became extinct in 1932 following the disappearance and presumed death of Booming Ben, the  final known member  of the subspecies. In the summer of 2014, non-profit organisation,  Revive & Restore  held a meeting with the community of Martha's Vineyard to announce their project to revive the heath hen in hopes of restoring and maintaining the sandplain grasslands. [ 76 ] [ 77 ]  On April 8th, 2020,  germs cells  were collected from greater prairie chicken eggs at  Texas A&M . [ 78 ] [ 79 ]


The  Yangtze giant softshell turtle  ( Rafetus swinhoei)  is a  softshell turtle  endemic to China and Vietnam and is possibly the largest living freshwater turtle. Due to various factors such as habitat loss, wildlife trafficking,  trophy hunting , and the  Vietnam War , the species population has been reduced to only three male individuals, rendering it functionally extinct similar to the northern white rhinoceros and ivory-billed woodpecker. [ 80 ] [ 81 ]  There is one captive individual in  Suzhou Zoo  in China, and two wild individuals at Dong Mo Lake in Vietnam. Efforts to save the species from extinction through various means of  assisted reproduction  in captivity have been ongoing since 2009 by the Suzhou Zoo and Turtle Survival Alliance.


Despite efforts to breed the turtles naturally, the eggs laid by the final known female were all  infertile  and unviable. In May 2015,  artificial insemination  was performed for the first time in the species. [ 82 ]  In July of the same year, the female laid 89 eggs, but like all previous natural attempts, they were all unviable. [ 83 ]  In April 2019, the female individual at the zoo died after another failed artificial insemination attempt. [ 84 ]  In 2020, a female was discovered in the wild, reigniting hope for the survival of the species. [ 85 ]  However, this individual was found dead in early 2023. [ 86 ]  Several searches across China and Vietnam are currently underway to locate female individuals to breed with the final known males, or to undergo artificial insemination. [ 87 ] [ 88 ] [ 89 ]


A "De-extinction Task Force" was established in April 2014 under the auspices of the  Species Survival Commission  (SSC) and charged with drafting a set of Guiding Principles on Creating Proxies of Extinct Species for Conservation Benefit to position the IUCN SSC on the rapidly emerging technological feasibility of creating a proxy of an extinct species. [ 90 ]


The  Judean date palm  is a species of  date palm  native to  Judea  that is estimated to have originally become extinct around the 15th century due to climate change and human activity in the region. [ 123 ]  In 2005, preserved seeds found in the 1960s excavations of  Herod the Great's  palace were given to Sarah Sallon by  Bar-Ilan University  after she came up with the initiative to germinate some ancient seeds. [ 124 ]  Sallon later challenged her friend, Elaine Solowey of the Center for Sustainable Agriculture at the  Arava Institute for Environmental Studies  with the task of germinating the seeds. Solowey managed to revive several of the provided seeds after hydrating them with a common household baby bottle warmer along with average fertiliser and growth hormones. [ 125 ]  The first plant grown was named after  Lamech's  father,  Methuselah , the oldest living man in the  Bible . In 2012, there were plans to  crossbreed  the male palm with what was considered its closest living relative, the  Hayani date  of Egypt to generate fruit by 2022. However, two female Judean date palms have been sprouted since then. [ 126 ]  By 2015 Methuselah had produced pollen that has been used successfully to pollinate female date palms. In June 2021, one of the female plants, Hannah, produced dates. The harvested fruits are currently being studied to determine their properties and  nutritional values . [ 127 ]  The de-extinct Judean date palms are currently at a  Kibbutz  located in  Ketura ,  Israel . [ 128 ]


The  Rastreador Brasileiro  (Brazilian Tracker) is a large  scent hound  from  Brazil  that was bred in the 1950s to hunt  jaguars  and wild pigs. It was originally declared extinct and delisted by the  Fédération Cynologique Internationale  and  Confederação Brasileira de Cinofilia  in 1973 due to  tick-borne diseases  and subsequent poisoning from  insecticides  in attempt to get rid of the ectoparasites. [ 129 ]  In the early 2000s, a group named Grupo de Apoio ao Resgate do Rastreador Brasileiro (Brazilian Tracker Rescue Support Group) dedicated to reviving the breed and having it relisted by Confederação Brasileira de Cinofilia began work to locate dogs in Brazil that had genetics of the extinct breed to breed a purebred Rastreador Brasileiro. [ 130 ]  In 2013, the breed was de-extinct through  preservation breeding  from descendants of the final original members and was relisted by the  FCI . [ 131 ] [ 132 ]


The  Floreana giant tortoise  ( Chelonoidis niger niger ) is a subspecies of the  Galápagos tortoise  endemic to  Floreana Island ,  Ecuador  that is believed to have become extinct by 1850 due to overexploitation, predation, and  habitat degradation  by sailors and invasive species such as  feral   livestock , rodents, and  stray dogs  and  cats . A deliberate  wildfire  started by Thomas Chappel, a crew member of the  Essex  in 1820 is also cited as a reason for the subspecies initial decline. [ 133 ]  In 2012, Floreana and  Volcán Wolf  tortoise  hybrids  were discovered  Isabela Island . [ 134 ]  Allegedly, these tortoises were imported or abandoned on the island in the early 19th century, allowing them to hybridise with the native subspecies. In 2017, a breeding programme was established to revive the subspecies through back breeding the hybrids to regain their genetic purity. [ 135 ]  As of 2025, 400 Floreana giant tortoises have been hatched on  Santa Cruz Island  with plans to release them into the wild on Floreana Island after the successful  extirpation  of invasive species. [ 136 ] [ 137 ]  However,  IUCN  has yet to update the status of the subspecies due to lack of a genetically pure specimen and the de-extinct subspecies has yet to reproduce naturally in the wild. [ 138 ]


In 2010, Sarah Sallon of  Arava Institute for Environmental Studies  grew a seed found in excavations of a cave in the northern  Judean desert  in 1986. The specimen, Sheba reached maturity in 2024 and is believed to be an entirely new species of  Commiphora  with many believing that she may be the  tsori  or Judean balsam, plants that are said to have healing properties in the  Bible . [ 139 ] [ 140 ]


The  Montreal melon , also known as the Montral market muskmelon, Montreal nutmeg melon, and in  French  as melon de Montréal  (Melon of/from Montreal)  is a cultivar of  melon  native to Canada and traditionally grown around the  Montreal  area. Despite its status as a delicacy on the east coast of North America, the Montreal melon disappeared from farms and was presumed extinct by the 1920s due to urbanisation in the region and being ill-suited for  agribusiness . In 1996, seeds of the lost melon were discovered in a  seed bank  in the American state of  Iowa . Since then, the plant has been reintroduced to its former range by local gardeners. [ 141 ] [ 142 ]


The  Dickey–Wicker Amendment  is the name of an appropriation bill  rider  attached to a bill passed by  United States Congress  in 1995, and signed by former President  Bill Clinton , which prohibits the  United States Department of Health and Human Services  (HHS) from using appropriated funds for the creation of human embryos for research purposes or for research in which human embryos are destroyed. HHS funding includes the funding for the  National Institutes of Health  (NIH). It is named after  Jay Dickey  and  Roger Wicker , two Republican Representatives. Technically, the Dickey Amendment is a rider to other legislation, which amends the original legislation. The rider receives its name from the name of the Congressman that originally introduced the amendment, Representative Dickey. The Dickey amendment language has been added to each of the Labor, HHS, and Education appropriations acts for fiscal years 1997 through 2009. The original rider can be found in Section 128 of P.L. 104–99. [ 1 ]


The wording of the rider is generally the same year after year. For fiscal year 2009, the wording in Division F, Section 509 of the Omnibus Appropriations Act, 2009,
 [ 2 ]  (enacted March 11, 2009) prohibits HHS, including NIH, from using fiscal year 2009 appropriated funds as follows:


In March 2009, President Obama issued an executive order which removed the restriction against federal funding of stem cell research. However, the Dickey–Wicker Amendment remains an obstacle for federally funded researchers seeking to create their own stem cell lines. [ 3 ] [ 4 ]  In August 2010, as part of preliminary motions in  Sherley vs Sebelius , Judge  Royce C. Lamberth  granted an injunction against federally funded embryonic stem cell (ESC) research on the grounds that the guidelines for ESC research "clearly violate" the Dickey–Wicker Amendment. In September 2010, he refused to lift the injunction pending the conclusion of the case and the issuance of his ruling and a likely appeal. In response, the Obama Justice Department asked the  U.S. Court of Appeals for the District of Columbia Circuit  to lift the injunction via an order pending the appeal of Judge Lamberth's ruling, [ 5 ]  which it did on April 29, 2011. [ 6 ]  Judge Lamberth was thereby obliged to reverse his ruling, and grudgingly dismissed the case entirely on July 27, 2011. [ 7 ]


In the 2–1 opinion of April 29, 2011, the appeals panel said that the Dickey–Wicker Amendment was "ambiguous" and that the National Institutes of Health had "reasonably concluded" that although federal funds could not be used to directly destroy an embryo, the amendment does not prohibit funding a research project using embryonic stem cells. This is an important distinction under the law, because for federal funds to be used directly to support the destruction of embryos—as opposed to indirect use just in embryo stem cell research that avoids killing the embryo—is supposedly a violation of the  Hyde Amendment , which has been ruled constitutional and which prohibits abortions using federal tax dollar funds (those questions will now have to be settled by the whole Court of Appeals for the District of Columbia Circuit sitting en banc, or perhaps, ultimately, by the  Supreme Court of the United States ).


Federal concern with  human embryo  research began over 25 years ago with the advent of  assisted reproduction technologies , i.e.  in vitro fertilization  (IVF) or " test tube babies ." [ 8 ]


Although the first report of laboratory studies of human fertilization appeared in Science in 1944, (the work was conducted in Brookline, Massachusetts), clinical IVF was successful first in  Great Britain  in 1978 for couples with infertility. [ 9 ]  IVF became standard of care in the United States in the early 1980s. As with all forms of clinical treatment, the medical community looked to basic science research to improve the safety and efficacy of IVF for mothers and babies. [ 8 ]


In 1979, an Ethics Advisory Board for the  National Institutes of Health  issued guidelines for research on early human embryos, but no action was taken. The Federal Policy for the Protection of Human Subjects (see:  Human subject research legislation in the United States ) enacted in 1977 remained in place: 45CFR § 46.204(d), "No application or proposal involving human in vitro fertilization may be funded by the Department or any component thereof until the application or proposal has been reviewed by the Ethical Advisory Board and the Board has rendered advice as to its acceptability from an ethical standpoint." [ 10 ]  Since there was no Ethics Advisory Board, federally funded research was not possible. [ 8 ]


Dolly  (5 July 1996 – 14 February 2003) was a female  Finn-Dorset  sheep and the first  mammal  that was  cloned  from an adult  somatic cell .  She was cloned by associates of the  Roslin Institute  in Scotland, using the process of  nuclear transfer  from a cell taken from a  mammary gland .  Her cloning proved that a cloned organism could be produced from a mature cell from a specific body part. [ 2 ]  Contrary to popular belief, she was not the first animal to be cloned. [ 3 ]


The employment of adult somatic cells in lieu of  embryonic stem cells  for cloning emerged from the foundational work of  John Gurdon , who cloned  African clawed frogs  in 1958 with this approach. The successful cloning of Dolly led to widespread advancements within stem cell research, including the discovery of  induced pluripotent stem cells . [ 4 ]


Dolly lived at the Roslin Institute throughout her life and produced several lambs. [ 5 ]  She was euthanized at the age of six years due to a progressive lung disease. No cause which linked the disease to her cloning was found. [ 6 ]


Dolly's body was preserved and donated by the Roslin Institute in Scotland to the  National Museum of Scotland , where it has been regularly exhibited since 2003.


Dolly was  cloned  by  Keith Campbell ,  Ian Wilmut  and colleagues at the  Roslin Institute , part of the  University of Edinburgh , Scotland, and the biotechnology company PPL Therapeutics, based near  Edinburgh . The funding for Dolly's cloning was provided by PPL Therapeutics and the  Ministry of Agriculture . [ 7 ]  She was born on 5 July 1996. [ 5 ]  She has been called "the world's most famous sheep" by sources including  BBC News  and  Scientific American . [ 8 ] [ 9 ]


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


Dolly was born on 5 July 1996 and had three mothers: one provided the egg, another the DNA, and a third carried the cloned embryo to term. [ 10 ]  She was created using the technique of  somatic cell nuclear transfer , where the  cell nucleus  from an adult cell is transferred into an unfertilized  oocyte  (developing egg cell) that has had its cell nucleus removed. The hybrid cell is then stimulated to divide by an electric shock, and when it develops into a  blastocyst  it is implanted in a surrogate mother. [ 11 ]  Dolly was the first clone produced from a cell taken from an adult mammal. [ 12 ] [ 13 ]  The production of Dolly showed that genes in the nucleus of such a mature  differentiated  somatic cell are still capable of reverting to an embryonic  totipotent  state, creating a cell that can then go on to develop into any part of an animal. [ 2 ]


Dolly's existence was announced to the public on 22 February 1997. [ 1 ]  It gained much attention in the media. A commercial with Scottish scientists playing with sheep was aired on TV, and a special report in  Time  magazine featured Dolly. [ 7 ]   Science  featured Dolly as the  breakthrough of the year . Even though Dolly was not the first animal cloned, she received media attention because she was the first cloned from an adult cell. [ 14 ]


Dolly lived her entire life at the  Roslin Institute  in Midlothian. [ 15 ]  There she was bred with a  Welsh Mountain ram  and produced six lambs in total. Her first lamb, named Bonnie, was born in April 1998. [ 5 ]  The next year, Dolly produced twin lambs Sally and Rosie; further, she gave birth to triplets Lucy, Darcy and Cotton in 2000. [ 16 ]  In late 2001, at the age of four, Dolly developed  arthritis  and started to have difficulty walking. This was treated with  anti-inflammatory  drugs. [ 17 ]


On 14 February 2003, Dolly was  euthanised  because she had a progressive lung disease and severe arthritis. [ 6 ]  A  Finn Dorset  such as Dolly has a life expectancy of around 11 to 12 years, but Dolly lived 6.5 years. A post-mortem examination showed she had a form of lung cancer called  ovine pulmonary adenocarcinoma , also known as Jaagsiekte, [ 18 ]  which is a fairly common disease of sheep and is caused by the  retrovirus   JSRV . [ 19 ]  Roslin scientists stated that they did not think there was a connection with Dolly being a clone, and that other sheep in the same flock had died of the same disease. [ 6 ]  Such lung diseases are a particular danger for sheep kept indoors, and Dolly had to sleep inside for security reasons. [ 20 ]


Some in the press speculated that a contributing factor to Dolly's death was that she could have been born with a genetic age of six years, the same age as the sheep from which she was cloned. [ 21 ]  One basis for this idea was the finding that Dolly's  telomeres  were short, which is typically a result of the  aging  process. [ 22 ] [ 23 ]  The Roslin Institute stated that intensive health screening did not reveal any abnormalities in Dolly that could have come from advanced aging. [ 21 ]


In 2016, scientists reported no defects in thirteen cloned sheep, including four from the same cell line as Dolly. The first study to review the long-term health outcomes of cloning, the authors found no evidence of late-onset, non-communicable diseases other than some minor examples of osteoarthritis and concluded "We could find no evidence, therefore, of a detrimental long-term effect of cloning by SCNT on the health of aged offspring among our cohort." [ 24 ] [ 25 ]


After her death Dolly's body was preserved via taxidermy and is currently on display at the  National Museum of Scotland  in Edinburgh. [ 26 ]


After cloning was successfully demonstrated through the production of Dolly, many other large mammals were cloned, including pigs, [ 27 ] [ 28 ]   deer , [ 29 ]  horses [ 30 ]  and bulls. [ 31 ]  The attempt to clone  argali  (mountain sheep) did not produce viable embryos. The attempt to clone a  banteng  bull was more successful, as were the attempts to clone  mouflon  (a form of wild sheep), both resulting in viable offspring. [ 32 ]  The reprogramming process that cells need to go through during cloning is not perfect and embryos produced by nuclear transfer often show abnormal development. [ 33 ] [ 34 ]  Making cloned mammals was highly inefficient – in 1996, Dolly was the only lamb that survived to adulthood from 277 attempts. By 2014, Chinese scientists were reported to have 70–80% success rates cloning pigs, [ 28 ]  and in 2016, a Korean company, Sooam Biotech, was producing 500 cloned embryos a day. [ 35 ]  Wilmut, who led the team that created Dolly, announced in 2007 that the nuclear transfer technique may never be sufficiently efficient for use in humans. [ 36 ]


Cloning may have uses in preserving endangered species, and may become a viable tool for reviving  extinct species . [ 37 ]  In January 2009, scientists from the Centre of Food Technology and Research of Aragon in northern Spain announced the cloning of the  Pyrenean ibex , a form of wild mountain goat, which was officially declared extinct in 2000. Although the newborn ibex died shortly after birth due to physical defects in its lungs, it is the first time an extinct animal has been cloned, and may open doors for saving endangered and newly extinct species by resurrecting them from frozen tissue. [ 38 ] [ 39 ]


In July 2016, four identical clones of Dolly (Daisy, Debbie, Dianna, and Denise) were alive and healthy at nine years old. [ 40 ] [ 41 ]


Scientific American  concluded in 2016 that the main legacy of Dolly has not been cloning of animals but in advances into  stem cell  research. [ 42 ]  Gene targeting was added in 2000, when researchers cloned female lamb Diana from sheep DNA altered to contain the human gene for  alpha 1-antitrypsin .  The human gene was specifically activated in the ewe’s mammary gland, so Diana produced milk containing human  alpha 1-antitrypsin . [ 43 ]  After Dolly, researchers realised that ordinary cells could be reprogrammed to  induced pluripotent stem cells , which can be grown into any tissue. [ 44 ]


The first successful cloning of a  primate species  was reported in January 2018, using the same method which produced Dolly. Two identical clones of a  macaque  monkey,  Zhong Zhong and Hua Hua , were created by researchers in China and were born in late 2017. [ 45 ] [ 46 ] [ 47 ] [ 48 ]


In January 2019, scientists in China reported the creation of five identical cloned  gene-edited  monkeys, again using this method, and the gene-editing  CRISPR - Cas9  technique allegedly used by  He Jiankui  in creating the first ever gene-modified human babies  Lulu and Nana . The monkey clones were made in order to study several medical diseases. [ 49 ] [ 50 ]


In 2003, the Belgian artist  Dominique Goblet  published a short comic strip about Dolly the cloned sheep with the title: “2004 Apparition de Dolly dans la campagne anglaise” [ 51 ]


" Dolly The Sheep " was initially released on November 13, 2012, as a  flash game  developed by the small game development company  Pozirk Games , in which Dolly the cloned sheep is being chased by evil scientists. For some time the game was available to play online as well as on mobile devices. As of June 14, 2023, it is only available online for desktop/laptop computers. [ 52 ]


The  egg cell  or  ovum  ( pl. :  ova ) is the  female   reproductive  cell, or  gamete , [ 1 ]  in most  anisogamous  organisms (organisms that  reproduce sexually  with a larger, female gamete and a smaller, male one). The term is used when the female gamete is not capable of movement (non- motile ). If the male gamete ( sperm ) is capable of movement, the type of sexual reproduction is also classified as  oogamous . A nonmotile female gamete formed in the oogonium of some algae, fungi, oomycetes, or bryophytes is an  oosphere . [ 2 ]  When fertilized, the oosphere becomes the  oospore . [ clarification needed ]


When egg and sperm fuse during  fertilisation , a  diploid  cell (the  zygote ) is formed, which rapidly grows into a new organism.


While the non-mammalian animal egg was obvious, the doctrine  ex ovo omne vivum  ("every living [animal comes from] an egg"), associated with  William Harvey  (1578–1657), was a rejection of  spontaneous generation  and  preformationism  as well as a bold assumption that mammals also reproduced via eggs.  Karl Ernst von Baer  discovered the mammalian ovum in 1827. [ 3 ] [ 4 ]  The fusion of spermatozoa with ova (of a starfish) was observed by  Oskar Hertwig  in 1876. [ 5 ] [ 6 ]


In animals, egg cells are also known as  ova  (singular  ovum , from the  Latin  word  ovum  meaning ' egg '). [ 7 ]  The term  ovule  in animals is used for the young ovum of an animal. In vertebrates, ova are produced by female  gonads  (sex glands) called  ovaries . A number of ova are present at birth in  mammals  and mature via  oogenesis .


Studies performed on humans, dogs, and cats in the 1870s suggested that the production of  oocytes  (immature egg cells) stops at or shortly after birth. A review of reports from 1900 to 1950 by zoologist  Solomon Zuckerman  cemented the belief that females have a finite number of oocytes that are formed before they are born. This dogma has been challenged by a number of studies since 2004. Several studies suggest that  ovarian stem cells  exist within the mammalian ovary. Whether or not mature mammals can actually create new egg cells remains uncertain and is an ongoing research question. [ 8 ] [ 9 ]


In all  mammals , the ovum is fertilized inside the female body. Human ova grow from primitive germ cells that are embedded in the substance of the  ovaries . [ 10 ]


The ovum is one of the largest  cells  in the human body, typically visible to the naked eye without the aid of a  microscope  or other magnification device. [ 11 ]  The human ovum measures approximately 120 μm (0.0047 in) in diameter. [ 12 ]


Ooplasm is like the yolk of the ovum, a cell substance at its center, which contains its  nucleus , named the  germinal vesicle , and the  nucleolus , called the  germinal disc . [ 13 ]


The ooplasm consists of the  cytoplasm  of the ordinary animal cell with its  spongioplasm  and  hyaloplasm , often called the  formative yolk ; and the  nutritive yolk  or  deutoplasm , made of rounded granules of fatty and  albuminoid  substances imbedded in the cytoplasm. [ 13 ]


Mammalian  ova contain only a tiny amount of the nutritive yolk, for nourishing the  embryo  in the early stages of its development only. In contrast, bird eggs contain enough to supply the chick with nutriment throughout the whole period of incubation. [ 13 ]


In the  oviparous  animals (all  birds , most  fish ,  amphibians  and  reptiles ), the ova develop protective layers and pass through the  oviduct  to the outside of the body. They are fertilized by male sperm either inside the female body (as in birds), or outside (as in many fish). After fertilization, an embryo develops, nourished by nutrients contained in the egg. It then hatches from the egg, outside the mother's body. See  egg  for a discussion of eggs of oviparous animals.


The egg cell's  cytoplasm  and  mitochondria  are the sole means the egg can reproduce by mitosis and eventually form a  blastocyst  after fertilization.


There is an intermediate form, the  ovoviviparous  animals: the embryo develops within and is nourished by an egg as in the oviparous case, but then it hatches inside the mother's body shortly before birth, or just after the egg leaves the mother's body. Some fish, reptiles and many  invertebrates  use this technique.


Nearly all land plants have alternating  diploid  and  haploid  generations. Gametes are produced by the haploid generation, which is known as the  gametophyte . The female gametophyte produces structures called  archegonia , and the egg cells form within them via  mitosis . The typical  bryophyte  archegonium consists of a long neck with a wider base containing the egg cell. Upon maturation, the neck opens to allow sperm cells to swim into the archegonium and fertilize the egg. The resulting zygote then gives rise to an embryo, which will grow into a new diploid individual, known as a  sporophyte . In  seed plants , a structure called the  ovule  contains the female gametophyte. The gametophyte produces an egg cell. After  fertilization , the ovule develops into a  seed  containing the embryo. [ 14 ]


In  flowering plants , the female gametophyte (sometimes referred to as the embryo sac) has been reduced to just eight cells inside the  ovule . The gametophyte cell closest to the  micropyle  opening of the ovule develops into the egg cell. Upon  pollination , a pollen tube delivers sperm into the gametophyte and one sperm nucleus fuses with the egg nucleus. The resulting zygote develops into an embryo inside the ovule. The ovule, in turn, develops into a  seed  and in many cases, the  plant ovary  develops into a  fruit  to facilitate the  dispersal  of the seeds. Upon  germination , the embryo grows into a  seedling . [ 14 ]


In the  moss   Physcomitrella patens , the  Polycomb  protein FIE is expressed in the unfertilised egg cell (Figure, right) as the blue colour after  GUS staining  reveals. Soon after fertilisation the FIE gene is inactivated (the blue colour is no longer visible, left) in the young embryo. [ 15 ]


In  algae , the egg cell is often called oosphere. [ citation needed ]   Drosophila  oocytes develop in individual egg chambers that are supported by nurse cells and surrounded by somatic follicle cells. The nurse cells are large polyploid cells that synthesize and transfer RNA, proteins, and organelles to the oocytes. This transfer is followed by the programmed cell death (apoptosis) of the nurse cells. During oogenesis, 15 nurse cells die for every oocyte that is produced. [ 16 ]  In addition to this developmentally regulated cell death, egg cells may also undergo apoptosis in response to starvation and other insults. [ 16 ]


In the context of  microbiology ,  enucleation  refers to removing the  nucleus  of a  cell  and replacing it with a different  nucleus . This is used mainly in  cloning  but can also be used for creating  hybrids  of plants or animals. It was discovered that cells can be chemically enucleated with  Cytochalasin B  in 1967. [ 1 ]


In  bioethics , the  ethics of cloning  concerns the  ethical  positions on the practice and possibilities of  cloning , especially  of humans . While many of these views are  religious  in origin, some of the questions raised are faced by  secular  perspectives as well.  Perspectives on human cloning are theoretical, as human therapeutic and reproductive cloning are not commercially used; animals are currently cloned in laboratories and in livestock production.


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


Opponents of cloning have concerns that technology is not yet developed enough to be safe, and that it could be prone to abuse, either in the form of clones raised as slaves, or leading to the generation of humans from whom organs and tissues would be harvested. Opponents have also raised concerns about how cloned individuals could integrate with families and with society at large.


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


Cloning of animals is opposed by animal-groups due to the number of cloned animals that suffer from malformations before they die, and while  meat  of cloned animals has been approved by the US FDA, its use is opposed by some other groups concerned about  food safety .


Chemical


Neurological


The various forms of  cloning , particularly  human cloning , are controversial. [ 1 ]  There have been numerous demands for all progress in the human cloning field to be halted. Most scientific, governmental and religious organizations oppose reproductive cloning. The  American Association for the Advancement of Science  (AAAS) and other scientific organizations have made public statements suggesting that human reproductive cloning be banned until safety issues are resolved. [ 2 ]  Serious  ethical concerns  have been raised by the future possibility of harvesting organs from clones. [ 3 ]


Advocates of human  therapeutic cloning  believe the practice could provide genetically identical cells for  regenerative medicine , and tissues and organs for transplantation. [ 4 ]  Such cells, tissues, and organs would neither trigger an immune response nor require the use of  immunosuppressive drugs . Both basic research and therapeutic development for serious diseases such as  cancer ,  heart disease , and  diabetes , as well as improvements in burn treatment and reconstructive and  cosmetic surgery , are areas that might benefit from such new technology. [ 5 ]  One bioethicist,  Jacob M. Appel  of  New York University , has gone so far as to argue that "children cloned for therapeutic purposes" such as "to donate bone marrow to a sibling with leukemia" may someday be viewed as heroes. [ 6 ] [ 7 ]


Proponents claim that human  reproductive cloning  also would produce benefits to couples who cannot otherwise procreate. In the early 2000s  Severino Antinori  and  Panos Zavos  stirred controversy when they publicly stated plans to create a fertility treatment that allows parents who are both infertile to have children with at least some of their DNA in their offspring. [ 8 ]


In  Aubrey de Grey 's proposed  SENS  (Strategies for Engineered Negligible Senescence), one of the considered options to repair the cell depletion related to  cellular senescence  is to grow replacement tissues from stem cells harvested from a cloned embryo. [ 9 ]


There are also ethical objections.  Article 11 of  UNESCO 's  Universal Declaration on the Human Genome and Human Rights  asserts that the reproductive cloning of human beings is contrary to human dignity, [ 10 ]  that a potential life represented by the embryo is destroyed when embryonic cells are used, [ 11 ]  and there is a significant likelihood that cloned individuals would be biologically damaged, due to the inherent unreliability of cloning technology. [ 12 ]


Ethicists have speculated on difficulties that might arise in a world where human clones exist. For example, human cloning might change the shape of  family  structure by complicating the role of parenting within a family of convoluted kinship relations. For example, a female DNA donor would be the clone's genetic twin, rather than mother, complicating the genetic and social relationships between mother and child as well as the relationships between other family members and the clone. [ 13 ]  In another example, there may be expectations that the cloned individuals would act identically to the human from which they were cloned, which could infringe on the right to self-determination. [ 14 ]


Proponents of  animal rights  argue that non-human animals possess certain moral rights as living entities and should therefore be afforded the same ethical considerations as human beings. This would negate the exploitation of animals in scientific research on cloning, cloning used in food production, or as other resources for human use or consumption. [ 15 ]


Religious views of cloning are mixed. [ 16 ] [ 17 ]


Hinduism  views on cloning are very diverse. While some Hindu people view therapeutic cloning as necessary to fix childlessness, others believe it is immoral to tamper with nature. [ 18 ]  The Sanatan Dharm (meaning the eternal set of duties for humans, which is what many people refer to Hinduism as) approves therapeutic cloning but does not approve human cloning. In Hinduism, one view has the creator, or the  Brahman  not as insecure to lay restrictions on scientific endeavours. Another view restricts human cloning. In  Jainism , the birth of Mahavira is depicted as an operation of embryo transfer. [ 19 ]


In modern-day India, there have been clones of livestock species. Examples include Garima from the  National Dairy Research Institute  located in  Karnal , where many other clones have been developed in Bovine species. [ citation needed ]


Jewish  view on cloning is unclear, but some orthodox rabbis allows cloning as a method of reproduction if no other method is available. Also Jewish religion treats all life equally even if it was formed by cloning. [ 20 ]  Liberal Jewish groups oppose the cloning of humans. [ 21 ]


Most of the Christian churches, including World council of Churches and United Methodist Church, oppose the research of cloning of either human embryos or whole human. [ 21 ]   The Roman Catholic Church , under the papacy of  Benedict XVI , condemned the practice of human cloning, in the magisterial instruction  Dignitas Personae , stating that it represents a "grave offense to the dignity of that person as well as to the fundamental equality of all people." [ 22 ]   Many conservative Christian groups have opposed human cloning and the cloning of human embryos, since they believe that life begins at the moment of  conception . [ 23 ]  Other  Christian  denominations such as the  United Church of Christ  do not believe a fertilized egg constitutes a living being, but still they oppose the cloning of embryonic cells.


There are different views when it comes to cloning in Islam, some scholars are of the view that human reproductive cloning is absolutely forbidden whilst others are of the view that there are some exceptions.


Animal cloning  is allowed in Islam only if it bring benefits to all people and no harm is caused to the animal used in the cloning process. [ 21 ] [ 24 ]


Cloned animals are used in medical research,  pet cloning  or for food. [ 25 ] [ 26 ]


In 2005, an article in  The Hastings Center Report  said:


Critics of pet cloning typically offer three objections: (1) the cloning process causes animals to suffer; (2) widely available pet cloning could have bad consequences for the overwhelming numbers of unwanted companion animals; and, (3) companies that offer pet cloning are deceiving and exploiting grieving pet owners. [ 27 ]


On December 28, 2006, the  U.S. Food and Drug Administration  (FDA) approved the consumption of meat and other products from cloned animals. [ 28 ]  Cloned-animal products were said to be indistinguishable from the non-cloned animals. Furthermore, companies would not be required to provide  labels  informing the consumer that the meat comes from a cloned animal. In 2007, some meat and dairy producers did propose a system to track all cloned animals as they move through the food chain, suggesting that a national database system integrated into the National Animal Identification System could eventually allow food labeling. [ 29 ] [ 30 ]  However, as of 2013 no tracking system exists, and products from cloned animals are sold for human consumption in the United States. [ 31 ] [ 32 ]


Critics have raised objections to the FDA's approval of cloned-animal products for human consumption, arguing that the FDA's research was inadequate, inappropriately limited, and of questionable scientific validity. [ 33 ] [ 34 ]   Several consumer-advocate groups are working to encourage a tracking program that would allow consumers to become more aware of cloned-animal products within their food. [ 35 ]


A 2013 review noted that there is widespread misunderstanding about cloned and cattle, and found that cloned cattle that reached adulthood and entered the food supply were substantially equivalent to conventional cattle with respect to the quality of meat and milk, and with respect to their reproductive capability. [ 36 ]


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. [ 37 ] [ 38 ]  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. [ 39 ]


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Fosmids  are similar to  cosmids  but are based on the  bacterial   F-plasmid . The cloning vector is limited, as a host (usually  E. coli ) can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. [ 1 ]  The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library.
The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex  genomes . Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. [ 2 ]  Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence. [ 3 ]


The fertility plasmid or  F-plasmid  was discovered by  Esther Lederberg  and encodes information for the biosynthesis of sex pilus to aid in bacterial conjugation. Conjugation involves using the sex pilus to form a bridge between two bacteria cells; this bridge allows the F+ cell to transfer a single-stranded copy of the plasmid so that both cells contain a copy of the plasmid. On the way into the recipient cell, the corresponding DNA strand is synthesized by the recipient. The donor cell maintains a functional copy of the plasmid. It later was discovered that the F factor was the first  episome  and can exist as an independent plasmid making it a very stable vector for cloning. Conjugation aids in the formation of bacterial clone libraries by ensuring all cells contain the desired fosmid. [ 4 ]


Fosmids are DNA vectors that use the F-plasmid origin of replication and partitioning mechanisms to allow cloning of large DNA fragments. A library that provides 20–70-fold redundant coverage of the genome can easily be prepared. [ 5 ]


The first step in sequencing entire genomes is cloning the genome into manageable units of some 50-200 kilobases in length.  It is ideal to use a fosmid library because of its stability and limitation of one plasmid per cell.  By limiting the number of plasmids in the cells the potential for recombination is decreased, thus preserving the genome insert. [ 6 ]


Fosmids contain several functional elements:


The methods of cutting and inserting DNA into fosmid vectors have been perfected.  There are now many companies that can create a fosmid library from any sample of DNA in a very short period of time at a relatively low cost.  This has been vital in allowing researchers to sequence numerous genomes for study.  Through a variety of methods, more than 6651 organisms genomes have been fully sequenced, with 58,695 ongoing. [ 7 ]


Sometimes it is difficult to accurately distinguish individual chromosomes based on chromosome length, arm ratio, and C-banding pattern.  Fosmids can be used as reliable cytological markers for individual chromosome identification and fluorescent in situ hybridization based metaphase chromosome karyotypes can be used to show whether the positions of these fosmids were successfully constructed. [ 8 ]


The fosmid system is excellent for rapidly creating chromosome-specific mini-BAC libraries from flow-sorted chromosomal DNA.  The major advantage of Fosmids over other cosmid systems lies in its capability of stably propagating human DNA fragments. [ 9 ]   Highly repetitive in nature, human DNA is well known for its extreme instability in multicopy vector systems.  It has been found that the stability increases dramatically when the human DNA inserts are present in single copies in recombination deficient  E. coli  cells. Therefore, Fosmids serve as reliable substrates for large scale genomic DNA sequencing. [ 2 ]


A  frozen zoo  is a storage facility in which genetic materials taken from animals (e.g.  DNA ,  sperm ,  eggs ,  embryos  and live tissue) are stored at very low temperatures (−196 °C) in tanks of liquid nitrogen. [ 1 ]  Material preserved in this way can be stored indefinitely [ 2 ]  and used for  artificial insemination ,  in vitro fertilization , embryo transfer, and  cloning . There are a few frozen zoos across the world that implement this technology for conservation efforts. Several different species have been introduced to this technology, including the Pyrenean ibex, Black-footed ferret, and potentially the white rhinoceros.


The first frozen zoo was established at the  San Diego Zoo  by pathologist  Kurt Benirschke  in 1972. [ 3 ] [ 4 ] [ 5 ]  At the time there was no technology available to make use of the collection, but Benirschke believed such technology would be developed in the future. [ 6 ]  The frozen zoo idea was later supported in  Gregory Benford 's 1992 paper proposing a Library of Life. [ 7 ]  Zoos such as the San Diego Zoo and research programs such as the Audubon Center for Research of Endangered Species [ 8 ] [ 2 ]  cryopreserve genetic material in order to protect the diversity of the gene pool of endangered species, or to provide for a prospective reintroduction of such extinct species as the  Tasmanian tiger [ 9 ]  and the  mammoth . [ 10 ]


Gathering material for a frozen zoo is rendered simple by the abundance of  sperm  in males. Sperm can be taken from an animal following death. The production of eggs, which in females is usually low, can be increased through  hormone treatment  to obtain 10–20  oocytes , dependent on the species. Some frozen zoos prefer to fertilize eggs and freeze the resulting embryo, as embryos are more resilient under the cryopreservation process. [ 11 ]  Some centers also collect skin cell samples of endangered animals or extinct species. The  Scripps Research Institute  has successfully made skin cells into cultures of special cells called induced pluripotent stem cells ( IPS cells ). It is theoretically possible to make sperm and egg cells from these IPS cells. [ 4 ]


Several animals whose cells were preserved in frozen zoos have been cloned to increase the  genetic diversity  of endangered species, as of 2021 [update] . One attempt to clone an extinct species was made in 2003; the newborn  Pyrenean ibex  died of a development disorder which may have been linked to the cloning, and there are not enough genetic samples in frozen zoos to re-create a breeding Pyrenean ibex population.


The Frozen Zoo at the San Diego Zoo's Institute for Conservation Research currently stores a collection of 8,400 samples from over 800 species and subspecies. [ 12 ]  It has acted as a forebear to similar projects at other zoos in the United States and Europe. [ 13 ] [ 14 ]  However, there are still less than a dozen frozen zoos worldwide. [ 2 ]


At the United Arab Emirates'  Breeding Centre for Endangered Arabian Wildlife (BCEAW) in Sharjah , the embryos stored include the extremely endangered  Gordon's wildcat  ( Felis silvestris gordoni ) and the  Arabian leopard  ( Panthera pardus nimr ) (of which there are only 50 in the wild). [ 11 ]


The Audubon Center for Research of Endangered Species, affiliated with the  University of New Orleans , is maintaining a frozen zoo. In 2000 the Center implanted a frozen-thawed embryo from the highly endangered African wildcat into the uterus of a domestic house cat, resulting in a healthy male wildcat. [ 15 ]


The  Frozen Ark  is a frozen zoo established in 2004 and jointly managed by the  Zoological Society of London , the  London Natural History Museum , and the  University of Nottingham . [ 16 ] [ 17 ]  This organization operates as a charity with many different departments including the DNA laboratory, consortium, taxon expert groups, and the database. [ 18 ]  In the DNA laboratory, samples are contained after collection from scientists, and different research projects are conducted there. The consortium acts as a bridge to bring together different, but important, groups from zoos, aquariums, museums, and universities. The taxon expert groups monitor the major phyla and lists like the IUCN Red List. The database is the essential piece as it holds all reports and records needed to perform all of the other functions for the charity. [ 18 ]  The hope for the future is for zoos and aquariums to be able to collect samples from their threatened and/or endangered species in house to help with conservation efforts. [ 18 ]  The collection and freezing of these samples allows for the distribution of gametes among populations. Samples can be collected from living hosts and from deceased hosts as well. [ 18 ] [ 19 ]


The  University of Georgia 's Regenerative Bioscience Center is building a frozen zoo. RBC Director Steven Stice and animal and dairy science assistant professor Franklin West created the facility with the thought of saving endangered cat species. The scientists have already extracted cells from a  Sumatran tiger , which could be used for artificial insemination. Artificial insemination provides a remedy for animals who, due to anatomical or physiological reasons, are unable to reproduce in the natural way. Reproduction of stored genetic material also allows for the fostering of genetic improvements, and the prevention of inbreeding. Modern technology allows for genetic manipulation in animals without keeping them in captivity. However, the success of their restoration into the wild would require the application of new science and a sufficient amount of previously collected material. [ 11 ]