gene manipulation techniques in animals
Experimental animals have been used in biomedical research for decades. In many cases, aspects of physiology and biochemistry have been investigated, and artificial manipulations have often been confined to examining the effect of altering the animal's environment or some aspect of its phenotype. The genetic engineering of animals is considerably more complicated, but it has a far reaching potential. Many of the modifications which have been carried out so far are aimed at producing larger specimens of certain animals, so that the breeding programs produce more food for the same space and effort. It is also possible to produce farm animals which are more resistant against diseases, some of which can spread from farm to farm and cause huge losses in stock and capital. It is expected that commercially available genetically modified animal products will be authorized for sale in the near future. Some animals, notably Drosophila and mice, have been particularly amenable to genetic analyses and traditional genetic manipulation of animals has involved carefully selected breeding experiments or exposure of animals to powerful chemical or radioisotopic mutagens. A new era in animal research was ushered in during the early 1980s when successful experiments designed to genetically modify animals by inserting foreign DNA were first reported. These new methodologies were expected to have many advantages for research but two major areas have benefited:
In order to create genetically modified animals, it is necessary to modify the DNA of germline cells so that the modified DNA is heritable. As a result, certain cells that have the capacity to differentiate into the different cells of an adult animal (or at least to give rise to germ line cells) were considered to be the optimal targets for introducing foreign DNA. The fertilized oocyte is one such cell, being totipotent. Other target cells are cells of very early stage embryos, including embryonic stem (ES) cells. Although such cells are postzygotic they represent a stage in development where there has been incomplete separation of thesoma and the germline. Such cells are therefore capable of giving rise to both somatic and germline cells. When a foreign DNA molecule is artifically introduced into the cells of an animal, a transgenic animal is produced. The foreign DNA molecule is called a transgene and may contain one or many genes. By inserting a transgene into a fertilized oocyte or cells from the early embryo, the resulting transgenic animal may be able to transmit the foreign DNA stably in its germline. Many different types of transgenic animals have been created including transgenic Drosophila, transgenic frogs, transgenic fish and a variety of transgenic mammals including mice, rats and various livestock animals. The technology of transgenesis and its applications are considered. Although transgenes often integrate into the host chromosomes without affecting the expression of any endogenous genes, occasionally the integration event alters endogenous gene expression (insertional mutation), producing a recognizable phenotype. This constitutes a form of in vivo mutagenesis, albeit at an unselected target gene. Gene targeting was developed as a method of in vivo mutagenesis in which the mutation is introduced into a preselected endogenous gene. This can be achieved in somatic cells, but gene targeting in cultured ES cells is particularly powerful because it can lead to the construction of an animal in which all nucleated cells contain a mutation at the desired locus. In mammals, gene targeting has been possible only in mice but research on ES cells from other species may extend the capacity for gene targeting in the near future. Note that in some cases gene targeting is used to produce a subtle mutation and as a result the ES cells used for blastocyst implantation do not contain any foreign sequences. The resulting mice may be described as genetically modified but not as transgenic.The ability to produce transgenic mice and particularly the ability to perform specific changes in a predetermined gene by gene targeting has permitted the design of many new animal models of human disease. Another experimental approach involving genetic manipulation of animals has had a major impact recently. In 1997 a new era in mammalian genetics was heralded when the procedure of somatic cell nuclear transfer permitted ‘cloning’ of an adult mammal for the first time. This involved transfer of the nucleus from an adult cell into an enucleated oocyte and the technology has subsequently been applied as an alternative route to generating transgenic animals.
- Gene function. While the use of cultured cells and cell extracts can be extremely valuable in studying gene expression and function, the ability to insert genes into whole animals or to selectively delete or alter single predetermined genes in an animal provides enormous power in studying gene function.
- Animal models of disease. Nature has provided some animal models of disease and some have been generated by random mutagenesis programmes, but not in a predetermined way. The new technologies held the promise of altering at will even single genes within a living animal in such a way as to mimic mutations faithfully in an analogous gene in humans, thereby providing a higher chance of resembling human disease phenotypes.
In order to create genetically modified animals, it is necessary to modify the DNA of germline cells so that the modified DNA is heritable. As a result, certain cells that have the capacity to differentiate into the different cells of an adult animal (or at least to give rise to germ line cells) were considered to be the optimal targets for introducing foreign DNA. The fertilized oocyte is one such cell, being totipotent. Other target cells are cells of very early stage embryos, including embryonic stem (ES) cells. Although such cells are postzygotic they represent a stage in development where there has been incomplete separation of thesoma and the germline. Such cells are therefore capable of giving rise to both somatic and germline cells. When a foreign DNA molecule is artifically introduced into the cells of an animal, a transgenic animal is produced. The foreign DNA molecule is called a transgene and may contain one or many genes. By inserting a transgene into a fertilized oocyte or cells from the early embryo, the resulting transgenic animal may be able to transmit the foreign DNA stably in its germline. Many different types of transgenic animals have been created including transgenic Drosophila, transgenic frogs, transgenic fish and a variety of transgenic mammals including mice, rats and various livestock animals. The technology of transgenesis and its applications are considered. Although transgenes often integrate into the host chromosomes without affecting the expression of any endogenous genes, occasionally the integration event alters endogenous gene expression (insertional mutation), producing a recognizable phenotype. This constitutes a form of in vivo mutagenesis, albeit at an unselected target gene. Gene targeting was developed as a method of in vivo mutagenesis in which the mutation is introduced into a preselected endogenous gene. This can be achieved in somatic cells, but gene targeting in cultured ES cells is particularly powerful because it can lead to the construction of an animal in which all nucleated cells contain a mutation at the desired locus. In mammals, gene targeting has been possible only in mice but research on ES cells from other species may extend the capacity for gene targeting in the near future. Note that in some cases gene targeting is used to produce a subtle mutation and as a result the ES cells used for blastocyst implantation do not contain any foreign sequences. The resulting mice may be described as genetically modified but not as transgenic.The ability to produce transgenic mice and particularly the ability to perform specific changes in a predetermined gene by gene targeting has permitted the design of many new animal models of human disease. Another experimental approach involving genetic manipulation of animals has had a major impact recently. In 1997 a new era in mammalian genetics was heralded when the procedure of somatic cell nuclear transfer permitted ‘cloning’ of an adult mammal for the first time. This involved transfer of the nucleus from an adult cell into an enucleated oocyte and the technology has subsequently been applied as an alternative route to generating transgenic animals.
The creation and applications of transgenic animalS.
Of the different transgenic animals that have been made thus far, transgenic Drosophila, transgenic frogs, and transgenic fish have been very important for understanding aspects of gene function and development in these species. Transgenic sheep and other transgenic livestock animals have been produced largely to serve as bioreactors, whole-animal expression cloning systems in which introduced genes are expressed to give large amounts of therapeutic or commercially valuable gene products. But it has been transgenic mice that have been the most useful to biomedical research, both in providing animal models of disease and in permitting the most useful analyses of mammalian gene function.
1. Transgenic animals can be produced following transfer of cloned DNA into fertilized oocytes and cells from very early stage embryos: Transgenesis involves transfer of foreign DNA into totipotent or pluripotent embryo cells (either fertilized oocytes, cells of the very early embryo or cultured embryonic stem cells) followed by insertion of the transferred DNA into host chromosomes. If the foreign DNA integrates into the chromosomes of a fertilized oocyte, the developing animal will be fully transgenic since all nucleated cells in the animal should contain the transgene. If chromosomal integration occurs later, at a post zygotic stage, the animal will be a mosaic, with some cells containing the transgene and some others lacking it. If the transgene is present in germline cells it can be passed through sperm or egg cells into some of the animal's progeny, and PCR-based tests can be used to quickly screen for the presence of the transgene. Progeny that are transgene positive can be expected to be fully transgenic; all their cells should have developed from a fertilized oocyte containing the transgene.
Pronuclear microinjection: To obtain transgenic mice by this route, females are super ovulated, mated to fertile males and sacrificed the next day. Fertilized oocytes are recovered from excised oviducts. The DNA of interest is then microinjected using a micromanipulator into the male pronucleus of individual oocytes. Surviving oocytes are reimplanted into the oviducts of foster females and allowed to develop into mature animals.
During this procedure, the microinjected DNA (transgene) randomly integrates into chromosomal DNA, usually at a single site, although rarely two sites of integration are found in a single animal. Individual insertion sites typically contain multiple copies of the transgenes integrated into chromosomal DNA as head-to-tail concatemers (it is not unusual to find 50 or more copies at a single insertion site). As a result of chromosomal integration, the transgenes can be passed on to subsequent generations in mendelian fashion: if the foreign DNA has integrated at the one-cell stage, it should be transmitted to 50% of the offspring.
Transfer into pre- or post implantation embryos Cells from very early stage embryos may be totipotent or at least pluripotent and can provide a route for foreign DNA to enter the germline: DNA can be transferred into unselected cells of very early embryos, as described in this section or into cell lines derived from embryonic stem cells.
One method that allows foreign DNA to integrate into the chromosomes of the target cells uses retroviruses, RNA viruses which naturally undergo an intermediate DNA form prior to integrating into cellular genomes. Infection of pre implantation mouse embryos with a retrovirus such as Moloney murine leukemia virus or injection of the retrovirus into early post implantation mouse embryos results in mosaic offspring. Retroviruses should integrate rarely and at random into accessible cells, and the use of replication- defective retroviruses provides heritable markers for clonal descendants of the target cell (unlike wild-type viruses which spread from cell to cell). This approach has been used, therefore, for studying cell lineage using reporter genes.
In Drosophila, efficient chromosomal integration is possible by using sequences from a Drosophila transposable element known as the P element to permit insertion of single copies of a gene at random in the genome. The gene or DNA fragment to be inserted is first manipulated so as to be flanked by the two terminal sequences of the P element. The modified DNA is then microinjected into a very young Drosophila embryo along with a separate plasmid containing a gene encoding a transposase. In the presence of the transposase the terminal P element sequences allow the intervening DNA fragment to transpose and as a result of the ensuing transposition events, the injected DNA often enters the germline in a single copy.
2. Cultured embryonic stem (ES) cells provide a powerful route to genetic modification of the germline: The microinjection of foreign DNA into fertilized oocytes is technically difficult and not suited to large-scale production of transgenic animals or to sophisticated genetic manipulation. A popular alternative, but one which has so far been restricted to the construction of genetically modified mice, involves transferring the foreign DNA initially into cultured embryonic stem(ES) cells. Mouse ES cells are derived from 3.5–4.5 day post coitum embryos and arise from the inner cell mass of the blastocyst. The ES cells can be cultured in vitro and retain the potential to contribute extensively to all of the tissues of a mouse, including the germline, when injected back into a host blastocyst and reimplanted in a pseudo pregnant mouse.
The developing embryo is a chimera: it contains two populations of cells derived from different zygotes, those of the blastocyst and the implanted ES cells. If the two strains of cells are derived from mice with different coat colors, chimeric offspring can easily be identified. Use of genetically modified ES cells results in a partially transgenic mouse. Because the injected ES cells can form all or part of the functional germ cells of the chimera, it is possible to derive fully transgenic mice. This is usually accomplished by screening the offspring of matings between chimeras (usually males) and mice with a coat color recessive to that of the strain from which the ES cells were derived.
The big advantage of ES cells is that they can be grown readily in culture. This means that a variety of genetic manipulations can be conducted in cultured ES cells. Importantly, the desired genetic modification can be verified in tissue culture, before injecting the genetically modified cells into a blastocyst prior to implantation. For example, the desired gene can be ligated to a marker gene, such as the neo gene, enabling a positive selection for cells that have been successfully transfected. The presence of the desired gene can also be verified quickly by a PCR-based assay. ES cells also offer the huge advantage of gene targeting by homologous recombination, a method which can permit a programed selective alteration of a single predetermined gene and also highly specific ways of chromosome engineering. Such approaches are extremely powerful for understanding gene function and also for creating animal models of disease.
The ES cell approach to constructing transgenic mice was made possible by the successful establishment in the early 1980s of stable cell lines from isolated mouse ES cells. ES cells were not so readily identifed in other mammals, although there have been some important recent successes.
3. Transgenic animals have been used for a variety of studies investigating mammalian gene expression and function: Transgenic animals have been extremely important for analyzing human genes and have helped greatly in our understanding of a variety of fundamental biological processes, notably in immunology, neurobiology, cancer and developmental studies. The following list is far from exhaustive but is intended to illustrate some major types of application:
1. Transgenic animals can be produced following transfer of cloned DNA into fertilized oocytes and cells from very early stage embryos: Transgenesis involves transfer of foreign DNA into totipotent or pluripotent embryo cells (either fertilized oocytes, cells of the very early embryo or cultured embryonic stem cells) followed by insertion of the transferred DNA into host chromosomes. If the foreign DNA integrates into the chromosomes of a fertilized oocyte, the developing animal will be fully transgenic since all nucleated cells in the animal should contain the transgene. If chromosomal integration occurs later, at a post zygotic stage, the animal will be a mosaic, with some cells containing the transgene and some others lacking it. If the transgene is present in germline cells it can be passed through sperm or egg cells into some of the animal's progeny, and PCR-based tests can be used to quickly screen for the presence of the transgene. Progeny that are transgene positive can be expected to be fully transgenic; all their cells should have developed from a fertilized oocyte containing the transgene.
Pronuclear microinjection: To obtain transgenic mice by this route, females are super ovulated, mated to fertile males and sacrificed the next day. Fertilized oocytes are recovered from excised oviducts. The DNA of interest is then microinjected using a micromanipulator into the male pronucleus of individual oocytes. Surviving oocytes are reimplanted into the oviducts of foster females and allowed to develop into mature animals.
During this procedure, the microinjected DNA (transgene) randomly integrates into chromosomal DNA, usually at a single site, although rarely two sites of integration are found in a single animal. Individual insertion sites typically contain multiple copies of the transgenes integrated into chromosomal DNA as head-to-tail concatemers (it is not unusual to find 50 or more copies at a single insertion site). As a result of chromosomal integration, the transgenes can be passed on to subsequent generations in mendelian fashion: if the foreign DNA has integrated at the one-cell stage, it should be transmitted to 50% of the offspring.
Transfer into pre- or post implantation embryos Cells from very early stage embryos may be totipotent or at least pluripotent and can provide a route for foreign DNA to enter the germline: DNA can be transferred into unselected cells of very early embryos, as described in this section or into cell lines derived from embryonic stem cells.
One method that allows foreign DNA to integrate into the chromosomes of the target cells uses retroviruses, RNA viruses which naturally undergo an intermediate DNA form prior to integrating into cellular genomes. Infection of pre implantation mouse embryos with a retrovirus such as Moloney murine leukemia virus or injection of the retrovirus into early post implantation mouse embryos results in mosaic offspring. Retroviruses should integrate rarely and at random into accessible cells, and the use of replication- defective retroviruses provides heritable markers for clonal descendants of the target cell (unlike wild-type viruses which spread from cell to cell). This approach has been used, therefore, for studying cell lineage using reporter genes.
In Drosophila, efficient chromosomal integration is possible by using sequences from a Drosophila transposable element known as the P element to permit insertion of single copies of a gene at random in the genome. The gene or DNA fragment to be inserted is first manipulated so as to be flanked by the two terminal sequences of the P element. The modified DNA is then microinjected into a very young Drosophila embryo along with a separate plasmid containing a gene encoding a transposase. In the presence of the transposase the terminal P element sequences allow the intervening DNA fragment to transpose and as a result of the ensuing transposition events, the injected DNA often enters the germline in a single copy.
2. Cultured embryonic stem (ES) cells provide a powerful route to genetic modification of the germline: The microinjection of foreign DNA into fertilized oocytes is technically difficult and not suited to large-scale production of transgenic animals or to sophisticated genetic manipulation. A popular alternative, but one which has so far been restricted to the construction of genetically modified mice, involves transferring the foreign DNA initially into cultured embryonic stem(ES) cells. Mouse ES cells are derived from 3.5–4.5 day post coitum embryos and arise from the inner cell mass of the blastocyst. The ES cells can be cultured in vitro and retain the potential to contribute extensively to all of the tissues of a mouse, including the germline, when injected back into a host blastocyst and reimplanted in a pseudo pregnant mouse.
The developing embryo is a chimera: it contains two populations of cells derived from different zygotes, those of the blastocyst and the implanted ES cells. If the two strains of cells are derived from mice with different coat colors, chimeric offspring can easily be identified. Use of genetically modified ES cells results in a partially transgenic mouse. Because the injected ES cells can form all or part of the functional germ cells of the chimera, it is possible to derive fully transgenic mice. This is usually accomplished by screening the offspring of matings between chimeras (usually males) and mice with a coat color recessive to that of the strain from which the ES cells were derived.
The big advantage of ES cells is that they can be grown readily in culture. This means that a variety of genetic manipulations can be conducted in cultured ES cells. Importantly, the desired genetic modification can be verified in tissue culture, before injecting the genetically modified cells into a blastocyst prior to implantation. For example, the desired gene can be ligated to a marker gene, such as the neo gene, enabling a positive selection for cells that have been successfully transfected. The presence of the desired gene can also be verified quickly by a PCR-based assay. ES cells also offer the huge advantage of gene targeting by homologous recombination, a method which can permit a programed selective alteration of a single predetermined gene and also highly specific ways of chromosome engineering. Such approaches are extremely powerful for understanding gene function and also for creating animal models of disease.
The ES cell approach to constructing transgenic mice was made possible by the successful establishment in the early 1980s of stable cell lines from isolated mouse ES cells. ES cells were not so readily identifed in other mammals, although there have been some important recent successes.
3. Transgenic animals have been used for a variety of studies investigating mammalian gene expression and function: Transgenic animals have been extremely important for analyzing human genes and have helped greatly in our understanding of a variety of fundamental biological processes, notably in immunology, neurobiology, cancer and developmental studies. The following list is far from exhaustive but is intended to illustrate some major types of application:
- Investigating gene expression and its regulation. Although evidence for cis-acting regulatory elements is often inferred initially from studies using cultured cells, they need to be validated in whole animal studies. Transgenes consisting of the presumptive regulatory sequence(s) coupled to a reporter gene, such as lacZ, provide a sensitive method of detecting gene expression and a powerful way of investigating regulation of gene expression. Long-range control of gene expression is often investigated using YAC transgenes.
- Investigating gene function by targeted gene inactivation. Specific genes can be inactivated by a gene targeting procedure to introduce a transgene into the target gene (insertional inactivation). The effect on the phenotype of creating a null mutation in the gene of interest can provide powerful clues to gene function.
- Investigating dosage effects and ectopic expression. In some cases, valuable information can be gained by over-expressing a transgene (e.g. Schedl et al., 1996) or by expressing it ectopically (the transgene is coupled to a tissue-specific promoter which causes expression in cells; the phenotypic consequence may provide valuable clues to function).
- Cell lineage ablation. Transgenes can be designed consisting of a tissue-specific promoter coupled to a sequence encoding a toxin, for example diphtheria toxin subunit A or ricin. When the promoter becomes active at the appropriate stage of tissue differentiation, the toxin is produced and kills the cells. Thus, certain cell lineages in the animal can be eliminated (cell ablation) and the phenotypic consequences monitored.
- Investigating gain of function. In principle, any mammalian gene that produces a dominant negative effect or gain of function can be investigated by introduction of an appropriate transgene. In some cases, this can provide proof of a suspected biological function. A classical example concerns the Sry gene. A variety of different genetic analyses had implicated this gene as a major male-determining gene but convincing proof was obtained using a transgenic mouse approach. The experiment consisted of transferring a cloned Sry gene into a fertilized 46,XX mouse oocyte. As a result of this artificial intervention, the resulting mouse, which nature had intended to be female, turned out to be male (Koopman et al., 1991).
- Modeling human disease. Insertional inactivation is often used to model loss of function mutations whilst gain of function mutations can often be modeled by inserting a mutant transgene.
Manipulating animals by somatic cell nuclear transfer
1. Principles and practice of animal cloning:
The term clones indicates genetic identity and so can describe genetically identical molecules (DNA clones), genetically identical cells or genetically identical organisms. Animal clones occur naturally as a result of sexual reproduction. For example, genetically identical twins are clones who happened to have received exactly the same set of genetic instructions from two donor individuals, a mother and a father. A form of animal cloning can also occur as a result of artificial manipulation to bring about a type of asexual reproduction. The genetic manipulation in this case uses nuclear transfer technology: a nucleus is removed from a donor cell then transplanted into an oocyte whose own nucleus has previously been removed. The resulting ‘renucleated’ oocyte can give rise to an individual who will carry the nuclear genome of only one donor individual, unlike genetically identical twins. The individual providing the donor nucleus and the individual that develops from the ‘renucleated’ oocyte are usually described as ‘clones’, but it should be noted that they share only the same nuclear DNA; they do not share the same mitochondrial DNA, unlike genetically identical twins.
Nuclear transfer technology was first employed in embryo cloning, in which the donor cell is derived from an early embryo, and has been long established in the case of amphibia. However, it was only comparatively recently when McGrath and Solter reported nuclear transplantation in the mouse embryo and paved the way for modern mammalian cloning. Subsequently, nuclear transplantation was conducted successfully in the eggs of domestic animals, including sheep and cows.
Unlike embryo cloning the prospect of cloning adults had seemed remote. During their development from (ultimately) the fertilized oocyte, adult somatic cells undergo an extensive series of cell division and differentiation steps. Until recently it was thought that these processes were accompanied by irreversible modifications to the genome. Even in frogs transplantation of an adult cell nucleus had never been reported to give rise to an adult animal; instead, the renucleated embryos underwent early development but thereafter failed to develop to term. A variety of early experiments in mice were also unsuccessful before the landmark study of Wilmut et al. 1997 reported successful cloning of an adult sheep. For the first time, an adult nucleus had been reprogrammed to become totipotent once more, just like the genetic material in the fertilized oocyte from which the donor cell had ultimately developed.
In the Wilmut et al. study, the donor cells were derived from a cell line established from adult mammary gland cells and were fused to an enucleated metaphase II-arrested oocyte. The donor cells were deprived of serum before use, forcing them to exit the cell cycle into a quiescent stage, Go (Stewart, 1997). A certain degree of gene silencing is a characteristic feature of the nuclei of Go cells. As egg cells are normally fertilized by transcriptionally inactive sperm cells, Gocells may be more amenable to full genetic reprogramming. Another consideration is the degree of chromosome condensation and of access to chromatin ‘remodeling factors’ such as transcription factors in the oocyte. In any event, the cloning was extremely inefficient: out of a total of 434 oocytes that were submitted to the procedure, only 29 developed to the transferable stage and of these only one developed to term, being born as the now famous Dolly. Subsequent doubts about the exact origin of the donor cell and whether Dolly really was an adult clone (as opposed to a contaminant fetal cell) have been allayed by genetic testing of Dolly and the adult mammary gland donor cells. Importantly, successful animal cloning has also been achieved by other groups with comparatively high success in cloning of adult mice and cows.
2. The successful cloning of an adult animal has major implications for research, medicine and society:
The report by Wilmut et al. (1997) has generated enormous attention, in the scientific and general press, both because of its novelty and the significance for future work. In particular, the possible extrapolation to cloning of humans has generated a great deal of controversy.
Basic research: Successful cloning of adult animals has forced us to accept that genome modifications once considered irreversible can be reversed and that the genomes of adult cells can be reprogammed by factors in the oocyte to make them totipotent once again. Research investigations into the control of gene expression during development and basic processes of somatic differentiation, somatic mutation, aging and repair processes will undoubtedly benefit from animal cloning, especially cloning of mice. Other more recent studies are now forcing us to reconsider the potency of other cells. For example, adult mouse neural stem cells transplanted into an irradiated host animal have very recently been shown to develop into a variety of blood cell types (myeloid, lymphoid and early hematopoietic cells) and so the developmental potential of stem cells is not restricted to the differentiated elements of the tissue in which they reside.
Cloning of livestock and transgenic animals: The successful cloning of adult sheep and cows is clearly attractive to people who wish to perpetuate prized livestock, racehorses, pets and endangered species. In addition, transgenic animals can be cloned. The traditional route for making a transgenic animal is by pronuclear microinjection. But this may be rather inefficient. Transgenic sheep and other livestock have been produced to serve as bioreactors, sources of medically valuable products such as human insulin. However, in the case of transgenic sheep, for example, only 2–4% of the founder animals born by implanting eggs which have been microinjected with a transgene turn out to be transgenic. Producing founder transgenic animals by nuclear transfer should be more efficient and will allow more sophisticated genetic modifications. An early success was achieved by Dr Wilmut's group who used fetal sheep cells containing a factor IX transgene as donor cells to generate transgenic sheep and this has been followed by cloning of transgenic cattle.
Human cloning: The most contentious issue in cloning animals is, of course, the potential extrapolation to cloning humans (Shapiro, 1997;Johnson, 1998). Clearly, the technology is still poorly developed and the comparatively high incidence of spontaneous abortions, perinatal losses and anomalous births observed in animal cloning would make the prospect of human cloning unappealing at present. In many countries, existing legislation would also preclude attempts at human cloning. For example, in the UK it is a criminal offence to experiment with human embryos without a licence, which will not be granted under any circumstances for experiments with embryos more than 14 days old.
Technological improvements in animal cloning will undoubtedly occur, however, and if the procedure were eventually to become both efficient and comparatively risk-free, there could be considerable pressure to apply nuclear replacement technology to human cells. Some applications need not involve human reproductive cloning. For example, nuclear replacement could be used to avoid transmission of inherited diseases derived from the mitochondria. Here, an unfertilized egg taken from an individual with mitochondrial disease could act as the donor with the nucleus being transferred into an enucleated egg from a donor containing normal mitochondria. The reconstructed egg could then be fertilized in vitro. The use of nuclear transfer technology for human reproductive cloning is, inevitably, more contentious. For some infertile couples or women, for example, it could provide a welcome method of having children. However, the expectation that could be placed on such a child could be damaging to that individual because the parent(s) and later the child may be especially conscious of genetic identity between individuals whose ages are quite different. Unlike identical twins whose development proceeds in parallel, for example, a cloned child could be only too aware of how he/she might develop in later life by observing a parent who was essentially genetically identical. Against this, many would argue that a person's character and capability is not determined exclusively be his/her genetic endowment; the environment also has a powerful role to play.
References: : http://www.ncbi.nlm.nih.gov/books/NBK7563/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078015/
http://www.geneticengineeringinhumans.com/ar/articles-on-genetic-engineering.php
http://www.nature.com/scitable/topicpage/genetic-inequality-human-genetic-engineering-768
The term clones indicates genetic identity and so can describe genetically identical molecules (DNA clones), genetically identical cells or genetically identical organisms. Animal clones occur naturally as a result of sexual reproduction. For example, genetically identical twins are clones who happened to have received exactly the same set of genetic instructions from two donor individuals, a mother and a father. A form of animal cloning can also occur as a result of artificial manipulation to bring about a type of asexual reproduction. The genetic manipulation in this case uses nuclear transfer technology: a nucleus is removed from a donor cell then transplanted into an oocyte whose own nucleus has previously been removed. The resulting ‘renucleated’ oocyte can give rise to an individual who will carry the nuclear genome of only one donor individual, unlike genetically identical twins. The individual providing the donor nucleus and the individual that develops from the ‘renucleated’ oocyte are usually described as ‘clones’, but it should be noted that they share only the same nuclear DNA; they do not share the same mitochondrial DNA, unlike genetically identical twins.
Nuclear transfer technology was first employed in embryo cloning, in which the donor cell is derived from an early embryo, and has been long established in the case of amphibia. However, it was only comparatively recently when McGrath and Solter reported nuclear transplantation in the mouse embryo and paved the way for modern mammalian cloning. Subsequently, nuclear transplantation was conducted successfully in the eggs of domestic animals, including sheep and cows.
Unlike embryo cloning the prospect of cloning adults had seemed remote. During their development from (ultimately) the fertilized oocyte, adult somatic cells undergo an extensive series of cell division and differentiation steps. Until recently it was thought that these processes were accompanied by irreversible modifications to the genome. Even in frogs transplantation of an adult cell nucleus had never been reported to give rise to an adult animal; instead, the renucleated embryos underwent early development but thereafter failed to develop to term. A variety of early experiments in mice were also unsuccessful before the landmark study of Wilmut et al. 1997 reported successful cloning of an adult sheep. For the first time, an adult nucleus had been reprogrammed to become totipotent once more, just like the genetic material in the fertilized oocyte from which the donor cell had ultimately developed.
In the Wilmut et al. study, the donor cells were derived from a cell line established from adult mammary gland cells and were fused to an enucleated metaphase II-arrested oocyte. The donor cells were deprived of serum before use, forcing them to exit the cell cycle into a quiescent stage, Go (Stewart, 1997). A certain degree of gene silencing is a characteristic feature of the nuclei of Go cells. As egg cells are normally fertilized by transcriptionally inactive sperm cells, Gocells may be more amenable to full genetic reprogramming. Another consideration is the degree of chromosome condensation and of access to chromatin ‘remodeling factors’ such as transcription factors in the oocyte. In any event, the cloning was extremely inefficient: out of a total of 434 oocytes that were submitted to the procedure, only 29 developed to the transferable stage and of these only one developed to term, being born as the now famous Dolly. Subsequent doubts about the exact origin of the donor cell and whether Dolly really was an adult clone (as opposed to a contaminant fetal cell) have been allayed by genetic testing of Dolly and the adult mammary gland donor cells. Importantly, successful animal cloning has also been achieved by other groups with comparatively high success in cloning of adult mice and cows.
2. The successful cloning of an adult animal has major implications for research, medicine and society:
The report by Wilmut et al. (1997) has generated enormous attention, in the scientific and general press, both because of its novelty and the significance for future work. In particular, the possible extrapolation to cloning of humans has generated a great deal of controversy.
Basic research: Successful cloning of adult animals has forced us to accept that genome modifications once considered irreversible can be reversed and that the genomes of adult cells can be reprogammed by factors in the oocyte to make them totipotent once again. Research investigations into the control of gene expression during development and basic processes of somatic differentiation, somatic mutation, aging and repair processes will undoubtedly benefit from animal cloning, especially cloning of mice. Other more recent studies are now forcing us to reconsider the potency of other cells. For example, adult mouse neural stem cells transplanted into an irradiated host animal have very recently been shown to develop into a variety of blood cell types (myeloid, lymphoid and early hematopoietic cells) and so the developmental potential of stem cells is not restricted to the differentiated elements of the tissue in which they reside.
Cloning of livestock and transgenic animals: The successful cloning of adult sheep and cows is clearly attractive to people who wish to perpetuate prized livestock, racehorses, pets and endangered species. In addition, transgenic animals can be cloned. The traditional route for making a transgenic animal is by pronuclear microinjection. But this may be rather inefficient. Transgenic sheep and other livestock have been produced to serve as bioreactors, sources of medically valuable products such as human insulin. However, in the case of transgenic sheep, for example, only 2–4% of the founder animals born by implanting eggs which have been microinjected with a transgene turn out to be transgenic. Producing founder transgenic animals by nuclear transfer should be more efficient and will allow more sophisticated genetic modifications. An early success was achieved by Dr Wilmut's group who used fetal sheep cells containing a factor IX transgene as donor cells to generate transgenic sheep and this has been followed by cloning of transgenic cattle.
Human cloning: The most contentious issue in cloning animals is, of course, the potential extrapolation to cloning humans (Shapiro, 1997;Johnson, 1998). Clearly, the technology is still poorly developed and the comparatively high incidence of spontaneous abortions, perinatal losses and anomalous births observed in animal cloning would make the prospect of human cloning unappealing at present. In many countries, existing legislation would also preclude attempts at human cloning. For example, in the UK it is a criminal offence to experiment with human embryos without a licence, which will not be granted under any circumstances for experiments with embryos more than 14 days old.
Technological improvements in animal cloning will undoubtedly occur, however, and if the procedure were eventually to become both efficient and comparatively risk-free, there could be considerable pressure to apply nuclear replacement technology to human cells. Some applications need not involve human reproductive cloning. For example, nuclear replacement could be used to avoid transmission of inherited diseases derived from the mitochondria. Here, an unfertilized egg taken from an individual with mitochondrial disease could act as the donor with the nucleus being transferred into an enucleated egg from a donor containing normal mitochondria. The reconstructed egg could then be fertilized in vitro. The use of nuclear transfer technology for human reproductive cloning is, inevitably, more contentious. For some infertile couples or women, for example, it could provide a welcome method of having children. However, the expectation that could be placed on such a child could be damaging to that individual because the parent(s) and later the child may be especially conscious of genetic identity between individuals whose ages are quite different. Unlike identical twins whose development proceeds in parallel, for example, a cloned child could be only too aware of how he/she might develop in later life by observing a parent who was essentially genetically identical. Against this, many would argue that a person's character and capability is not determined exclusively be his/her genetic endowment; the environment also has a powerful role to play.
References: : http://www.ncbi.nlm.nih.gov/books/NBK7563/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078015/
http://www.geneticengineeringinhumans.com/ar/articles-on-genetic-engineering.php
http://www.nature.com/scitable/topicpage/genetic-inequality-human-genetic-engineering-768
#Disclaimer: This website is for curriculum based purpose so not to be used for professional use.