22 October 2017
2. Criteria for Assessing...
3. Gene Transfer to Human Embryos
4. Sperm-mediated Gene Transfer
5. Episomal Vectors
6. Totipotent Cells
Int J Med Sci 2004; 1(2):76-91. doi:10.7150/ijms.1.76
Gene Therapy: The Potential Applicability of Gene Transfer Technology to the Human Germline
School of Contemporary Sciences, University of Abertay, Dundee, DD1 1HG, United Kingdom
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How to cite this article:
Smith KR. Gene Therapy: The Potential Applicability of Gene Transfer Technology to the Human Germline. Int J Med Sci 2004; 1(2):76-91. doi:10.7150/ijms.1.76. Available from http://www.medsci.org/v01p0076.htm
The theoretical possibility of applying gene transfer methodologies to the human germline is explored. Transgenic methods for genetically manipulating embryos may in principle be applied to humans. In particular, microinjection of retroviral vector appears to hold the greatest promise, with transgenic primates already obtained from this approach. Sperm-mediated gene transfer offers potentially the easiest route to the human germline, however the requisite methodology is presently underdeveloped. Nuclear transfer (cloning) offers an alternative approach to germline genetic modification, however there are major health concerns associated with current nuclear transfer methods. It is concluded that human germline gene therapy remains for all practical purposes a future possibility that must await significant and important advances in gene transfer technology.
Keywords: gene targeting, gene therapy, genetic modification, pronuclear microinjection, sperm-mediated gene transfer
Human germline genetic modification is theoretically possible: the technologies of animal transgenesis (pronuclear microinjection, sperm-mediated gene transfer, nuclear transfer, etc) could in principle be applied to humans. The purpose of this paper is to consider the potential for applying the available genetic modification (GM) technologies to the goal of achieving human germline gene therapy.
If germline gene therapy does become a technically viable proposition, one crucial question must be asked: Why do it? There is in effect a 'golden rule' applying to disorders potentially amenable to germline gene therapy: in any disorder with enough molecular knowledge available to allow the prospect of germline gene therapy, that same knowledge should also be sufficient to allow detection of the disease-causing sequences via embryo pre-screening. Given the low transfer efficiencies and safety risks available at present (i.e. extrapolating from animal transgenesis), candidate disorders would have to be severe and unavoidable by pre-screening. However, it is conceptually possible that gene transfer technologies improve to the point at which it becomes easier and safer to perform germline gene therapy than to carry out embryo pre-screening. In this futuristic scenario of expanded genetic knowledge coupled with effective gene transfer technology, germline GM might become the preferred therapeutic route.
The possibility of human germline genetic modification raises several important and vexing bioethical issues, including questions of responsibility towards future generations, difficulties of distinction between gene therapy and genetic enhancement, and the spectre of eugenics [1, 2, 3] . Thus, human germline genetic modification is far more ethically contentious than somatic gene therapy. However, such bioethical matters are beyond the scope of the present discussion. Instead, this paper focuses solely upon scientific issues related to human germline gene therapy.
2. Criteria for Assessing Applicability to Human Germline Gene Therapy
An ideal gene transfer system in the context of human germline gene therapy would have the following features: (a) the ability to deliver transgenes in a highly efficient manner; (b) non-prohibitive cost and expertise requirements; (c) minimal risk of causing insertional DNA damage; (d) low rate of mosaicism; (e) high DNA carrying capacity; (f) the ability to permit adequate and controlled transgene expression; and (g) the ability to target transgenes to precise genomic loci.
Unfortunately, no single system amongst the presently available systems is able to provide all of features (a-g) above. Indeed, some gene transfer systems are so thoroughly unsuited to human germline gene therapy that they are not considered here. Of the systems that offer some positive features, in every case major drawbacks exist. In each case, particular scientific advances are required before the methods would be suitable for use in human germline gene therapy. In this respect, methods that require a relatively small degree of scientific research should be seen as more plausible than methods requiring many years of progress towards distant (possibly unobtainable) goals.
3. Gene Transfer to Human Embryos
Most transgenic animals have been produced via the introduction of transgenes into embryos, and the associated technology and underpinning science is accordingly well developed. Thus, the human embryo is a potential candidate for human germline gene therapy.
Unless dramatic improvements in the technologies are forthcoming, certain transfection methods are not at present a realistic proposition for gene transfer into human zygotes. Such methods include liposome-mediated gene transfer, electroporation, naked DNA uptake and many viral vectors. The problem is one of low transfection frequencies, coupled with fact that zygotes must be harvested (as opposed to grown in vitro). This leaves pronuclear microinjection and retroviral transfer as the only contenders presently available that might be adapted for use with embryos in human germline gene therapy.
Jon Gordon in 1980 demonstrated that exogenous DNA could be introduced into the germline simply by the physical injection of a solution of cloned DNA into zygote pronulei  . Subsequently, pronuclear microinjection has become the most widely used method of germline gene transfer, despite the fact that it remains an intrinsically costly and laborious approach. The technique is most established with mice, however gene transfer via pronuclear microinjection has also been carried out with a wide range of other mammals including rats, rabbits, and farmyard animals. Accordingly, it is to be expected that the human zygote should in principle be similarly amenable to gene tranfer via pronuclear microinjection. The microinjection technique is intrinsically simple, although it requires expensive equipment and high levels of skill  . A fine glass needle is loaded with DNA solution. Under the microscope, the needle is guided through the cytoplasm towards one of the zygote's pronuclei. A nanolitre quantity of DNA solution is injected, bringing typically two hundred DNA molecules into the pronucleus.
Pronuclear microinjection would be an obvious choice of transgene delivery method for human embryos. The technique is well established in animals, and is likely to be directly applicable to the human zygote [6, 7, 8] . Zygotes from various mammalian species have particular characteristics that necessitate amendments to the basic (murine) technique. For example, bovine and porcine zygotes are optically opaque, due to the presence of lipid granules in the cytoplasm; this necessitates centrifugation to displace the obscuring cytoplasmic material such that the pronuclei become visible. Similarly, the pronuclei in ovine zygotes are very difficult to visualise, due to sharing a very similar refractive index with the cytoplasm; this necessitates the use of top-quality optics, such as differentiation interference contrast (DIC) microscopy, instead of standard phase contrast microscopy. Thus, empirical adjustments enable pronuclear microinjection to be employed with zygotes from essentially any mammal. It would be surprising and unfortunate if the human zygote proved to be an exception to this rule. Indeed, visualisation of the pronuclei in human fertilised eggs is not problematic.
Although pronuclear microinjection would probably be usable with human zygotes, the major inherent problems of the method render it less than ideal for human germline gene therapy. A major problem is the relatively low rate of transgene integration: in mice, the overall efficiency of transgenesis (taking into account embryo loss in vitro and in vivo) is typically ca. 2% [9, 10, 11] . This level of efficiency is perfectly practicable for animal transgenesis, but it would be problematic for humans. Moreover, murine pronuclear microinjection transgene uptake values are several times higher than those achieved with other (non-rodent) species. Accordingly, even with hormonal induction of superovulation, the numbers of zygotes available per woman would be a strongly limiting factor in the potential use of pronuclear microinjection for human germline gene therapy.
Embryo pre-screening (preimplantation genetic screening) might be one possible way around the problem of low transgene uptake efficiency. Using established techniques, one or two blastomeres could be taken from 8 cell stage embryos and analysed by PCR for the presence of transgene DNA. However, such pre-screening would not be 100% reliable, due to mosaicism within the early embryo. Following microinjection and successful integration of the transgene sequences, the transgene would be expected to be present in only 50% of the resulting blastomeres. Assuming that in humans, as with mice, 3 blastomeres are recruited to form the entire inner cell mass (ICM) [12, 13] , then 1 in 8 of the resulting individuals would contain no transgene sequences, another 1 in 8 would contain transgene sequences in 100% of their cells, and the remaining 6 from 8 individuals would be mosaics, consisting of 1/3rd or 2/3rd transgene-containing cells. Accordingly, pre-screening would have a failure rate of more than 50%. The only feasible way that pre-screening might work at an acceptable level of efficiency would be to screen blastocyst-stage embryos. However, blastocyst biopsy techniques are in their infancy, and it remains to be seen whether such techniques could be applied to ICM cells (as opposed to trophoblast cells).
Extrapolating from murine data, it would typically require ca. 50 zygotes to produce one genetically modified individual. Assuming 8 eggs per superovulation cycle, it would take approximately 6 months per woman to obtain 50 eggs. Pronuclear microinjection involving such a period of time, if coupled with effective blastocyst pre-screening to select for the small number of transgene–containing embryos, might be a feasible means of performing human germline gene therapy. However, reported pronuclear microinjection efficiency values are significantly lower for most mammals other than mice. If human pronuclear microinjection turned out to have a similar efficiency as that obtained with sheep or pigs, then the time taken per genetically modified individual would be ca. 5-fold longer – i.e. more than 2.5 years. And if the rate of transgenesis turned out to be similar to that obtained with cattle, the time would extend beyond 8 years. The efficiency of transgene uptake through pronuclear microinjection is simply not known for humans, nor can it be known a priori. Thus, a circular problem exists: only if the efficiency turned out to be fortuitously high (i.e. similar to murine rates) would there be any point in attempting the technique with humans – but the necessary data on efficiency could only come from actual attempts with humans.
Another problem associated with pronuclear microinjection concerns transgene expression. Only around 60% of pronuclear microinjection-derived mice show transgene expression. Furthermore, in the animals showing expression, there are frequently problems of low-level expression or inappropriate expression (e.g. non-tissue-specific, non-temporal). Accordingly, pronuclear microinjection as a means to human germline gene therapy requires improvements in transgene expression. It is the non-targeted nature of transgene integration associated with pronuclear microinjection that is the root cause of expression problems. Some improvements may come from advances in transgene design, such as the use of matrix attachment regions (MARs) or locus control regions (LCRs): placed on either side of a gene within a transgene construct, these 'insulator' sequences appear to allow the gene to occupy a separate chromosomal domain and thus avoid position-related expression problems  . However, the best solution would be to target transgenes to precise genomic loci, and at present this is not possible with pronuclear microinjection. Given the fact that even the best designs of targeting transgene undergo random integration more frequently than targeted integration, the only foreseeable way to achieve high efficiency gene targeting with pronuclear microinjection would be to stimulate homologous recombination (HR) by co-injecting appropriate recombinase enzymes with the transgene. However, elucidation of such enzymes is at an early stage, and it remains to be seem whether this approach could ever provide the quantum leap improvements in targeting efficiency that would be required in the case of human germline gene therapy.
Random integration also raises the concern that an endogenous gene will be damaged by transgene insertion. The degree of risk for any one insertion event must approximate to the proportion of coding sequences (plus controlling elements) within the human genome, a figure of no more than 2%. Thus, endogenous gene damage may be expected to occur in around 1 in every 50 human zygotes integrating transgene DNA. In embryos sustaining such damage, there are several possible outcomes: (a) where a developmentally crucial gene is damaged, the result is likely to be embryo death, and the subsequent non-appearance of a genetically modified individual; (b) where one allele of an important gene is affected, haplosufficiency may permit the development of a normal or near-normal genetically modified individual; (c) where a non-essential gene is affected (such as an allele for hair colour, or a repeated gene), the resulting genetically modified individual may contain a phenotypic change that has no health implications; or (d) where an important gene is affected, debility is likely to occur in the resulting genetically modified individual. Outcomes (a-c), while not desirable, would not necessarily be highly problematic, and the occurrence of these outcomes means that the undesirable outcome (d) would occur at a frequency significantly lower than 1 in 50 genetically modified individuals. Nevertheless, such magnitude of risk implies that pronuclear microinjection in its present stage of development is not acceptable as a means to human germline gene therapy.
The genome of retroviruses can be manipulated to carry exogenous DNA. Retroviral vectors (RVVs) are one of the most frequently employed forms of gene delivery in somatic gene therapy [15, 16, 17] . Additionally, RVVs are able to deliver genes to the germline, as established in animal transgenesis [18, 19, 20] . Zygotes may be incubated in media containing high concentrations of the resultant retroviral vector. Alternatively, retroviral vector-producing cell monolayers may be used, upon which zygotes are co-cultivated. In either case, up to ca. 90% of (surviving) embryos will be infected. Following zygote transfer into pseudopregnant females, the infected embryos should give rise to transgenic offspring. Molecular genetic analysis of transgenics produced in this way usually show integration of a single proviral copy into a given chromosomal site. Rearrangements of the host genome are normally restricted to short direct repeats at the site of integration. In many embryos the germline cells contain viral integrants: thus, transmission of the transgene to the next generation will often occur. Methods have also been developed to allow infection into postimplantation embryos. In this context, virus uptake is effective for many somatic cell lines, however germline cells are infected at low frequency, due to a high level of mosaicism [15, 16] .
Retroviral transfer remains an alternative to pronuclear microinjection in the context of human germline gene therapy. Traditional RVVs would be of minimal potential use, due to the high levels of mosaicism associated with these vectors. However, the new generation of lentiviral vectors would avoid such problems [21, 22, 23] . These vectors have the additional advantage of high gene transfer rates (70-80% of animals born are transgenic). Accordingly, lentiviral vectors represent plausible candidates for human germline gene therapy. However, the small insert capacity (9-10 kb) would preclude the transfer of many human genes. Additionally, control possibilities are less with RVV delivered transgenes compared with transgenes delivered by microinjection.
The safety problems associated with RVVs (insertional oncogenesis, viral reactivation) would also be a major concern [24, 25, 26] . In principle, judicious genetic alteration of the lentivirus genome would ensure that the resultant vector would have a very high level of safety. However, given the critical context of human germline gene therapy, one would have to question whether our basic scientific understanding of retroviruses is sufficiently advanced to empower rational vector design. Somatic gene therapy provides a salutary lesson here. Human trials involving several hundred patients have been carried out for over a decade using RVVs. Despite the theoretical risks referred to above, a lack of reports of serious adverse affects has resulted in a growing acceptance of the practical safety of RVVs. However, it has been recently reported that two patients (both young children) being treated for X-linked severe combined immunodeficiency disease (SCID-X) using RVV-based vectors have developed leukaemia. In both patients, RVV had integrated into a gene (LMO2) known to cause leukaemia if activated inappropriately. It is not known why the same endogenous gene had been targeted by the RVV concerned. The full cause of leukaemia in these patients is still under investigation, however the fact that both patients share the same integration site, coupled with the fact that the patients were both from the same (10-patient) trial, strongly implicates the particular RVVs employed in this trial [27, 28, 29] . Indeed, clinical trials involving this particular RVV-based therapy have been halted pending further investigations and pre-clinical trials  . It is to be hoped that enhanced RVV design will prevent any recurrence of iatrogenic leukaemia or similar serious adverse affects in somatic gene therapy. However, the occurrence of such adverse RVV effects lends weight to the argument that more basic virology is needed before any potential human germline gene therapy RVV could be deemed sufficiently safe. At the very least, extensive in vitro (cell culture) and in vivo (mammalian transgenesis) experimentation would be required in order to establish the safety of any proposed RVV (lentiviral-based or otherwise) for human germline gene therapy.
Microinjection of Retroviral Vector
A combination of microinjection with retroviral vectors has proved successful with bovines  and primates  . In the primate case, microinjection was used to deliver a retroviral vector into the perivitelline space of 224 mature rhesus oocytes. (The oocytes were subsequently fertilized by intracytoplasmic sperm injection.) The retroviral vector particles had an envelope type known to recognise and bind to the membrane of all cell types. The retroviral vector was microinjected at a developmental stage at which the oocyte nuclear membrane was absent, thus permitting nuclear entry. From 20 embryo transfers, three animals were born, one of which was transgenic. Additionally, a miscarried pair of twins was transgenic. Although this 'combined' method of gene transfer is laborious, it is the only approach that has permitted the generation of transgenic primates thus far.
Given the success with primates of microinjection of RVV into oocytes, this approach is likely to be effective for human germline gene therapy. The drawbacks would be similar to those associated with (a) microinjection (i.e. embryo loss) and (b) RVVs (i.e. transgene size limitations, problems with control of expression, safety risks). The process would also be laborious, but it would be expected to avoid the problems of mosaicism associated with most RVVs. Additionally, this form of gene transfer might require fewer eggs than required for pronuclear microinjection. The reported overall rate of transgenesis with rhesus monkeys was 1.3%; although this compares unfavourably with murine efficiencies (up to ca. 6% transgenic), it is significantly better than the rates achieved for animals such as sheep, cows and pigs. Moreover, this 'combined' technique is in its infancy, and its efficiency may well improve with use.
4. Sperm-mediated Gene Transfer
The scientific literature contains over forty reports of the successful in vitro uptake of exogene constructs (transgenes) by animal sperm cells [33, 34] . A majority of these reports provide evidence of post-fertilisation transfer and maintenance of transgenes. Several of the studies report the subsequent generation of viable progeny animals, the cells of which contain transgene DNA sequences. While a minority of studies have used 'augmentation' techniques (electroporation or liposomes) to 'force' sperm to capture exogenes, the standard methodology is very straightforward: prior to in vitro fertilisation (IVF) or artificial insemination (AI), 'washed' sperm cells are simply incubated in a DNA-containing solution. As a potential tool for genetically manipulating animals, sperm-mediated gene transfer (SMGT) has the advantages of simplicity and cost-effectiveness, in contrast with more established methods of transgenesis such as pronuclear micrinjection.
However, despite the above successes and regardless of its potential utility, SMGT has not yet become established as a reliable form of genetic modification. Concerted attempts to utilise SMGT have often produced negative results. The most notable example of such a failure is to be found in the collated results of several independent research groups: of 890 mice analysed, not a single animal contained transgene DNA  .
Indeed, some biologists have expressed scepticism of the fundamental basis for SMGT [36, 37] . Such scepticism is posited on the assumption that major evolutionary chaos would result if sperm cells were able to act as exogene vectors. Given that the reproductive tracts contain 'free' DNA molecules (originating from natural cell death and breakage), it seems reasonable to expect sperm cells to be highly resistant to the risk of picking up such molecules  .
Nevertheless, there exists a fairly well established body of empirical data showing that sperm cells are able, at least under particular experimental circumstances, to interact with and carry exogenes [39, 40] . Furthermore, isolated reports of the successful use of SMGT for genetic modification continue to be published. A notable recent example is the generation of several transgenic pigs following the artificial insemination of sows with sperm cells preincubated with transgene DNA [41, 42] .
There are two possible ways to make sense of the above experimental and theoretical considerations. The first possible explanation is that SMGT is fundamentally unattainable. If so, the empirical evidence in support of SMGT must be faulty. For example, perhaps sperm can associate with exogenous DNA but cannot convey the DNA into the oocyte; and transgene sequences may have been erroneously identified in tissue samples, perhaps due to DNA contamination affecting sensitive detection methods such as PCR. This scenario is certainly not impossible: scientific research contains several examples of theory being misled by mistaken data. Indeed, early reports of SMGT were compared with the (then contemporary) claims of “cold fusion” in physics  . By contrast, the second possible explanation is that SMGT is viable, and that the claims of experimental success were not made in error. In this case, the explanation for the successful results must be that certain favourable factors applied in the fortuitous cases in which transgenes were taken up and transferred by sperm. Accordingly, several researchers have made efforts to elucidate such hidden parameters.
Underpinning such research into hidden factors has been the notion of the existence of 'inhibitory' factors (IFs) associated with sperm cells. These IFs are envisaged to prevent exogenous DNA uptake so as to protect the genetic integrity of the conceptus. The corollary of this notion is that successful instances of sperm cells taking up exogenous DNA may be attributed to the fortuitous removal or inhibition of IF(s)  .
Seminal fluid reportedly contains an inhibitory factor (IF-1) that appears to actively block the binding of exogenous DNA to sperm and to the above-mentioned proteins  . Additionally, three classes of proteins identified in sperm cells have been claimed to exhibit DNA-binding properties [44, 45] . There is also some evidence that the binding of transgene DNA can trigger the activation of endogenous nucleases in sperm cells, which cleave both transgene and sperm chromosomal DNA [46, 47, 48] . The possible existence of IF(s) or other mechanisms against foreign DNA may explain the varied and often negative results obtained from attempts to use sperm to act as transgene vectors.
A superficial binding of exogenous DNA to sperm cells would be very unlikely to result in successful transgenesis, given the rigours of fertilisation. Conceptually, therefore, it is necessary to envisage the exogenous DNA being actively taken up by the sperm cell. Ultrastructural autoradiographic studies have indicated that exogenous DNA becomes concentrated within the posterior part of the nuclear area of the head, the inference being that binding of DNA by the sperm is followed by internalisation [49, 50] .
One very interesting possibility is the combination of naked DNA autouptake with microinjection, a process that has been termed 'transgenICSI'. In this recent approach, sperm exposed to naked transgene molecules are microinjected into oocytes. Success has been reported with mice, with approximately 20% of founder animals integrating and expressing the transgene [51, 52] . Transgene uptake and expression following transgenICSI has also been reported in rhesus monkey embryos [53, 54] and porcine embryos [55, 56] , although transgenic offspring did not result.
The success of transgenICSI provides support for the notion that sperm are indeed able to act as transgene vectors. However, some caution is required in making such a conclusion. Firstly, the experiments conducted need to be repeated and built upon before it can be said with certainty that the effect is a real one. Secondly, it could be the case that the trangene molecules bound only weakly to the sperm cells, such that only direct delivery (by ICSI) permitted the DNA to remain in place. If so, then this would not support the notion that SMGT can work when used with IVF or AI, because weakly bound or superficially located DNA might be stripped away and lost from the incoming pronucleus during fertilisation. If it were correct that ICSI is an indispensable part of the process, then SMGT would appear to have little advantage over pronuclear microinjection in terms of inherent technical difficulties and expense. However, the efficiency of the process does appear to be somewhat better than that of pronuclear microinjection. The available experimental data on standard human ICSI (i.e. not involving genetic modification) indicate that: (a) the majority (ca. 75%) of eggs are successfully fertilised; and (b) lysis following ICSI occurs at a relatively low rate (ca. 10%) [57, 58, 59] . For transgenICSI, the reported rates of success (i.e. transgenics per transfer) vary, but a figure of around ca. 35% is fairly typical [51, 52, 53, 54, 55, 56].
Whereas it would be somewhat surprising if sperm cells have the inherent ability to easily capture and transfer naked transgene molecules such that the DNA remains in place during fertilisation, it remains conceptually possible to use transfection techniques to 'force' sperm cells to capture (and thus transfer) exogenous DNA. Success has been claimed in this regard using electroporation and liposome-mediated gene transfer. Since 1990, several reports claiming successful transgene uptake and/or transfer following electroporation of sperm cells have been published, and there have been a number of reports of sperm cells taking up liposome-encapsulated DNA  . More research is clearly needed to determine whether and to what extent transfection techniques such as liposome-mediated gene transfer or electroporation may be able to augment SMGT. Nevertheless, given that these gene transfer techniques have been shown to work with a wide range of somatic cell types, in vitro and in vivo, there is no reason to presume that sperm cells are inherently unable to be transfected using such methods.
An alternative possibility could be to introduce the transgene into testicular (sperm) stem cells in vivo. This would in principle remove the need to collect, manipulate or transfer eggs, thus providing a major streamlining of germline GM. Preliminary results have been reported in mice, where transgene constructs were directly injected into the testis. For example, 60-70% of sperm were reported to carry the transgene following injection of naked DNA into the vas deferens  , with a follow-up report claiming detection of the transgene in the cells of 7.5% of offspring animals produced following fertilisation with the transgene-bearing sperm  . Similar results were reported by Sato et al, using liposome-encapsulated transgene molecules injected close to the epididymis [62, 63, 64] .
In vitro gene delivery into ex vivo spermatogonial stem cells of both adult and immature animals has recently been reported  . Nagano et al obtained stable transgene integration and expression in up to 20% of murine spermatogonial stem cells following retroviral transgene delivery  . Genetically modified stem cells were transferred into the testes of infertile recipient mice, leading to transgeneity in ca. 4.5% of the resultant progeny, plus successful transmission to subsequent generations. Similar results were obtained by Orwig et al in rats  . Although this form of transgenesis is at an early stage of development, preliminary work with spermatogonial stem cells in other mammals such as pigs and goats suggests that the approach is likely to be widely applicable [68, 69] . If human ex vivo spermatogonial stem cells are similarly able to pick up and transmit transgenes, an exciting potential route to germline gene therapy might emerge.
If SMGT does indeed work as reported, or if it can be made to work, then this would have very profound implications for human germline gene therapy. Gene transfer into embryos (using pronuclear microinjection or RVVs) is inherently very costly and technically demanding, due in large part to the need to remove embryos from the female, to manipulate the embryos and finally to return the embryos to the reproductive tract. By contrast, SMGT coupled with AI would permit germline GM with minimum levels of expense and expertise, as would SMGT coupled with testicular injections. Thus, SMGT would in principle permit the widespread use of human germline gene therapy: relatively poor countries would be able to use the technique, and highly centralised facilities would not be required. Of course, such easily available human germline gene therapy would raise serious ethical concerns.
However, even if SMGT were to prove effective as a means to gene transfer, it would be fundamentally limited in the context of human germline gene therapy due to its unsuitability as a means of gene targeting. This limitation is of course shared with the embryo-based gene transfer methods considered above. However, there are at least some glimmers of hope for future gene targeting possibilities in the case of embryo-based approaches: some (albeit very limited) success has been achieved with targeting RVVs  , and the low natural rate of HR in zygotes might conceivably be increased if appropriate recombinase enzymes were to be discovered and co-injected  . By contrast, there have been no reports of gene targeting using SMGT, and it is difficult to envisage even in outline how this might ever be achieved.
5. Episomal Vectors
Various extrachromosomal plasmid vectors (episomes) have been used as transgenes [72, 73] . Such vectors have been employed to produce transgenic animals, via a variety of routes including pronuclear microinjection and SMGT [74, 75, 76] . In the context of human germline gene therapy, such vectors offer the potential advantage of eliminating the threat to genome integrity associated with uncontrolled genomic integration. However, in transgenic animals, episomal plasmid vectors tend to behave in an unstable fashion [76, 77] . During development, plasmid copy numbers fluctuate and plasmids are lost from some cells. Plasmid inheritance to subsequent generations of animals is similarly problematic. Moreover, worrying health problems (such as tumour formation) have been associated with some episomal vectors  . Of course, the behaviour of an episome must relate in large part to its genetic constitution, and therefore stability problems and safety limitations may in principle be surmounted by improved plasmid design. However, until such improvements are realised, episomal plasmid vectors could not be considered for human germline gene therapy.
Autonomous artificial 'mini-chromosomes', (mammalian artificial chromosomes, MACs) have been constructed and successfully introduced into mammalian cells [79, 80] . MACs comprise centromeres, telomeres and replication origins, and are maintained autonomously within the host cell. Structural genes, promoters and enhancers (etc) can be included in MACs. Preliminary research indicates that MACs can be used, via pronuclear microinjection, to create transgenic animals in which the MACs are maintained autonomously  . In the context of human germline gene therapy, these specialised constructs would be expected to give a number of benefits compared with integrated transgenes, including higher and more controllable expression. More speculatively, MACs may be able to function as genetic 'platforms' for the safe subsequent receipt of incoming transgenes. Although this technology is in its infancy, MACs would appear to hold significant future potential for human germline gene therapy [81, 82, 83, 84, 85] .
6. Totipotent Cells
At present, gene targeting requires the use of in vitro selection in order to enrich for rare targeted cells amongst a majority of random integration cells. In vitro selection cannot be conducted on embryos or sperm cells. Consequently, gene targeting in the context of human germline gene therapy would require gene transfer to be carried out with some form of dividing cells in vitro.
Inner cell mass (ICM) cells from the mouse blastocyst can be propagated in vitro as embryonic stem (ES) cells [86, 87] . In contrast to other cultured cell lines, ESCs retain their normal karyotype even after many months in culture, during which time they remain totipotent. Furthermore, ESCs are capable of colonising the embryo. These unique properties allow ESCs to form chimeras when injected into blastocysts or aggregated with morulae. The resultant embryos can be transferred to the uterus of a pseudopregnant female mouse for gestation. In cases where an ESC has successfully contributed to the embryo, the resultant offspring will be chimeric (up to ca. 50% of animals). The ESC contribution to a mouse can high (up to ca. 80% of the cells), and will often include the germline cells. However, it should be noted that, with some transgenes, the production of chimeras can be problematic or even unattainable, especially when germline transmission is required to breed pure lines of heterozygous or homozygous animals [86, 87, 88, 89] .
It is during the in vitro culture stage that ESCs may be transgenically manipulated [90, 91] . Many gene delivery systems are effective with ESCs, including viral vectors, liposomes, and electroporation. The great advantage of ESCs is that they can be subjected to a range of selective agents in vitro, which allows the selection of particular transgenic modifications. This ability makes ESCs extremely useful for gene targeting experiments and applications  ,  .
However, the use of ESCs is limited due to the fact that, to date, the mouse is the only animal from which ESC lines have been unequivocally established. It would be surprising if this limitation represents a fundamental biological barrier. However, further empirical work is needed before totipotent ESC lines become available for other species. Indeed, efforts to isolate non-murine ESCs have been ongoing for nearly two decades but to date no germline-competent ESCs have been isolated in other vertebrates [94, 95] .
In 1998, Thomson et al isolated ESCs from human blastocysts  .
Subsequently many other researchers have also isolated human ESCs [97, 98, 99, 100] , and a new field in biology has resulted. Furthermore, gene targeting has been achieved in human ESCs  . However, totipotency has not been demonstrated in any human ESC line. Unfortunately, this may prove to be a rather intractable situation: proof of totipotency could only (given current technology) come from the establishment of a chimeric human being. It is manifest that the necessary experiments required to pursue this goal would be ethically unacceptable. Thus, the ESC route presently remains firmly closed against human germline gene therapy.
The successful transfer of 'reprogrammed' sheep donor nuclei has recently been achieved [102, 103, 104] . Unfertilised, metaphase-stage enucleated ('universal recipient') eggs received the transferred nuclei. Donor nuclei originated from somatic cells that had been forced into a form of cell cycle stasis (by incubating the cells in a minimal nutrient medium), such that DNA replication and gene expression were halted (or virtually so). Nuclear transfer was conducted by depositing a donor cell under the zona pellucida of a universal recipient egg, and fusing the two cells by electrical stimulation. This process resulted (in some cases) in successful embryo development, the donor nuclei having been 'reprogrammed' into totipotency. Offspring were produced following the transfer of such 'reconstructed' embryos to recipient ewes. Subsequent molecular genetic testing showed that the lambs' DNA had originated from the donor cells. In some of the experiments, the donor nuclei were obtained from embryo-derived cultured cell lines. Following these groundbreaking experiments, successful cloning from cultured cells of various mammals including cattle, goats and pigs has been reported [105, 106] . Interestingly, a human ESC line has recently been derived from cloned human blastocysts produced by NT  , pointing to a possible new field of application for NT technology.
The prospects for germline GM via NT are very significant: transgenes can be introduced to somatic donor cells in vitro, permitting germline genetic modifications. This has been achieved in animals  . Several gene delivery systems are applicable to NT, including liposomes and electroporation. Moreover, because selection can be applied to cultured donor cells, NT can be used to achieve germline gene-targeting. Gene targeted transgenic animals have been created in this way [108, 109] . Thus, NT is potentially able to provide the same range of transgenic manipulations presently available in mice (via the ESC route) to all mammal species. Accordingly, it seems probable that the technique could in principle be readily applied to humans as a means to achieving germline modifications.
However, in comparison with ESC transgenesis, NT has thus far proved to be relatively inefficient: only a small proportion of reconstructed embryos survive to become live animals. For example, McCreath et al produced live targeted sheep at an efficiency of less than 4%  , and Lia et al produced live targeted pigs at an efficiency of less than 2%  . Such low efficiencies, presuming reconstructed human embryos to behave similarly, represent a potential problem for human NT-based germline gene therapy. Although embryo pre-selection could be used to ensure that only transgene-containing embryos were allowed to gestate, the problem would remain that a large number of valuable donor eggs would be required for each GM attempt.
The health status of NT-derived animals is also proving to be problematic [111, 112] . Developmental abnormalities are very common, and frequently result in death (foetal or postnatal) or debility. For example, of fourteen live-born lambs, seven died within 30 hours of birth, and four died within twelve weeks  . Similarly, out of seven piglets, two piglets died shortly after birth, and one died at 17 days; only one appeared to be entirely free of developmental abnormalities  . Transgenesis and gene targeting are not of themselves implicated: the health problems are associated with NT per se. During the in vitro (cell culture) stage, the pattern of chromosomal imprinting may change; there are indications that inappropriate expression of imprinted genes following such epigenetic alteration may be mainly responsible for the poor health of NT-derived animals [113, 114, 115] . Research into epigenetic reprogramming in NT embryos is in progress, and it is to be hoped that developmental abnormalities arising from NT will eventually be eliminated or reduced in frequency. Meanwhile, it is anticipated that NT-related health problems, to the extent that the basis for such is epigenetic, are unlikely to affect the offspring of surviving first-generation animals. However, until such time as the first-generation health problems are solved, NT appears to be too dangerous to consider for human germline gene therapy. This is unfortunate, because without NT (or human ESCs) the in vitro selection required for germline gene targeting cannot be conducted. Thus, germline GM in humans would be restricted to 'add-in' alterations; gene knockout and gene repair germline alterations in humans are not a practical proposition with the technology available at present.
Non-selective gene targeting
In embryonic stem cells and in certain somatic cells in vitro, unusually high levels of gene targeting have been reported. Isogenic transgenes, derived from the same (syngenic) laboratory animal strain as the target animal, contain homology blocks that are genetically identical (or virtually identical) to the target homology regions. Riele et al  reported a 20-fold improvement in targeting efficiency when an isogenic transgene was used to target the retinoblastoma susceptibly gene (Rb) in murine ESCs, yielding a remarkably favourable ratio of random to targeted integration (approximately 1:4). Similar results were obtained from a systematic study by Vandeursen and Wieringa  , in which the creatine kinase M gene (CKM) in ESCs was targeted.
More recently, adeno-associated virus (AAV) vectors have been used to gene target somatic cells at high frequencies. Hirata et al used AAV vectors to introduce transgenes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts  . The transgenes were targeted at high frequencies, such that the majority of transgene-containing cells had undergone gene targeting with an appropriately designed vector. AAV targeting frequencies have been further improved by selective creation of double-strand DNA breaks in the target site [119, 120] . Most recently, adult human mesenchymal stem cells (MSCs) have also been targeted with high efficiency using AVV vectors  .
The foregoing reports suggest that in ESCs and in certain somatic cells the efficiency of gene targeting can be sufficient to bypass the need for selection. Selection-based gene targeting places limits on transgene design, due to the need to engineer the requisite selective elements into the transgene. Therefore, the ability to conduct gene targeting in somatic cells without the need for selection would be a welcome addition to the armamentarium of gene transfer technologies that may in future permit human germline gene therapy.
It is probable that the human germline could be readily manipulated using current transgenic techniques. To achieve this, pronuclear microinjection would probably be effective, as would retroviral transfer, particularly using lentivirus-based vectors. A combination of microinjection and RVVs would probably be most effective – indeed such a combination has recently given rise to the first transgenic primates. SMGT may be effective also, at least in some forms of the approach, such as transgenICSI. However, AI-based SMGT is not yet an established method of transgenesis, therefore the prospects for this potentially very important form of gene transfer are less certain. Totipotent human ESCs have not been established for humans, thus ESC-based gene transfer remains – despite its effectiveness in mice – unavailable for human germline gene therapy. The lack of human ESCs leaves NT-based gene transfer as the only method that might be able to permit gene targeting in human germline gene therapy. NT could probably be readily applied to humans; however, the high level of health problems observed in first generation NT-derived animals render the approach in its present form unfeasible for human germline gene therapy. Table 1 summarises the key features of the major candidate methods that might serve to achieve human germline gene therapy.
If human gene transfer technology is limited to adding-in gene functions via non-targeted transgene integration, and if the process needs be performed on individual embryos isolated from the reproductive tract, it is likely that human germline gene therapy will remain insufficiently safe, excessively inefficient and of inadequate clinical value to permit its use. The widespread availability and applicability of safe and effective human germline gene therapy would require the development of gene transfer methods that would (a) permit gene targeting while (b) avoiding the need for ex vivo embryo isolation and manipulation. Unfortunately, at present these two requirements are mutually exclusive. Laborious manipulations involving large numbers of embryos would in principle best be avoided by the use of SMGT, either in vivo or ex vivo. However, high-efficiency gene targeting is not available at present without the use of in vitro selection. Thus, the widespread use of human germline gene therapy does not appear likely to flow from incremental improvements in current GM methods. Rather, widespread human germline gene therapy would appear to be a future possibility that must await substantial scientific advancement. Naturally, it is impossible to predict when such improvements might be forthcoming.
Conflict of interest
The author has declared that no conflict of interest exists.
1. Smith KR. Gene therapy: Theoretical and bioethical concepts. Arch Med Res. 2003 ;34 (4) :247 -268
2. Resnik DB, Langer PJ. Human germline gene therapy reconsidered. Hum Gene Ther. 2001 ;12 (11) :1449 -1458
3. McDonough PG. The ethics of somatic and germline gene therapy. Adolescent Gynecology and Endocrinology- Basic and Clinical Aspects. 1997 ;816 :378 -382
4. Gordon JW, Scangos GA, Plotkin DJ. et al. Genetic transformation of mouse embryos by micro-injection of purified DNA. Proc Natl Acad Sci U S A. 1980 ;77 (12) :7380 -7384
5. Hogan B, Beddington R, Constantin F, Lacy E. Manipulating the mouse embryo. New York: Cold Spring Harbor Laboratory. 1994
6. Houdebine LM. The methods to generate transgenic animals and to control transgene expression. J Biotechnol. 2002 ;98 (2-3) :145 -160
7. Houdebine LM. Transgenesis and medical applications. Pathol Biol. 2002 ;50 (6) :380 -387
8. Wall RJ. Transgenic livestock: Progress and prospects for the future. Theriogenology. 1996 ;45 (1) :57 -68
9. Bagis H, Papuccuoglu S. Studies on the production of transgenic mice. Turk J Vet Anim Sci. 1997 ;21 (4) :287 -292
10. Page RL, Canseco RS, Russell CG. et al. Transgene detection during early murine embryonic-development after pronuclear microinjection. Transgenic Res. 1995 ;4 (1) :12 -17
11. Hirabayashi M, Takahashi R, Ito K. et al. A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes. Exp Anim. 2001 ;50 (2) :125 -131
12. Bishop JO. Transgenic Mammals. Harlow: Pearson Education. 1999
13. Gilbert SF. Developmental Biology. Sunderland, Mass: Sinauer Associates. 1997
14. Smith KR. Gene transfer in higher animals: theoretical considerations and key concepts. J Biotechnol. 2002 ;99 (1) :1 -22
15. Braas G, Searle PF, Slater NKH, Lyddiatt A. Strategies for the isolation and purification of retroviral vectors for gene therapy. Bioseparation. 1996 ;6 (4) :211 -228
16. Morgan RA, French Anderson W. Human Gene Therapy. Annu Rev Biochem. 1993 ;62 :191 -
17. Hu WS, Pathak VK. Design of retroviral vectors and helper cells for gene therapy. Pharmacol Rev. 2000 ;52 (4) :493 -511
18. Cepko CL, Roberts BE, Mulligan RC. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 ;37 (3) :1053 -1062
19. Jaenisch R, Jahner D, Nobis P. et al. Chromosomal position and activation of retroviral genomes inserted into the germ line of mice. Cell. 1981 ;24 (2) :519 -529
20. Gridley T, Soriano P, Jaenisch R. Insertional mutagenesis in mice. Trends Genet. 1987 ;3 (6) :162 -166
21. Ikawa M, Tanaka N, Kao WWY, Verma IM. Generation of transgenic mice using lentiviral vectors: A novel preclinical assessment of lentiviral vectors for gene therapy. Mol Ther. 2003 ;8 (4) :666 -673
22. Lois C, Hong EJ, Pease S. et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002 ;295 (5556) :868 -872
23. Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci U S A. 2002 ;99 (4) :2140 -2145
24. Cornetta K, Morgan RA, Anderson WF. Safety issues related to retroviral-mediated gene transfer in humans. Human Gene Ther. 1991 ;2 (1) :5 -14
25. Gunter KC, Khan AS, Noguchi P. The safety of retroviral vectors. Human Gene Ther. 1993 ;4 (5) :643 -
26. Temin HM. Safety considerations in somatic gene-therapy of human-disease with retrovirus vectors. Hum Gene Ther. 1990 ;1 (2) :111 -123
27. Buckley RH. Gene therapy for SCID - a complication after remarkable progress. Lancet. 2002 ;360 (9341) :1185 -1186
28. Kaiser J. Gene Therapy - Seeking the cause of induced leukemias in X-SCID trial. Science. 2003 ;299 (5606) :495 -495
29. Gansbacher B, Danos O, Dickson G. et al. French group reports on the adverse event in a clinical trial of gene therapy for X-linked severe combined immune deficiency (X-SCID) - Position statement from the European Society of Gene Therapy (ESGT). J Gene Med. 2003 ;5 (1) :82 -84
30. Fox JL. US authorities uphold suspension of SCID gene therapy. Nat Biotechnol. 2003 ;21 (3) :217 -217
31. Chan AWS, Homan EJ, Ballou LU. et al. Transgenic cattle produced by reverse-transcribed gene transfer in oocytes. Proc Natl Acad Sci U S A. 1998 ;95 (24) :14028 -14033
32. Chan AWS, Chong KY, Martinovich C. et al. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Science. 2001 ;291 (5502) :309 -312
33. Gandolfi F. Sperm-mediated transgenesis. Theriogenology. 2000 ;53 (1) :127 -137
34. Smith KR. Sperm cell mediated transgenesis: A review. Anim Biotechnol. 1999 ;10 (1-2) :1 -13
35. Brinster RL, Sandgren EP, Behringer RR, Palmiter RD. No simple solution for making transgenic mice. Cell. 1989 ;59 (2) :239 -241
36. Birnstiel ML, Busslinger M. Dangerous Liaisons - Spermatozoa as natural vectors for foreign DNA. Cell. 1989 ;57 (5) :701 -702
37. Chen TM, Chen YH. Transgenic sperm or deadly missiles?. Fertil Steril. 1996 ;66 (1) :167 -167
38. Smith KR. The role of sperm-mediated gene transfer in genome mutation and evolution. Med Hypotheses. 2002 ;59 (4) :433 -437
39. Zani M, Lavitrano M, French D, Lulli V. The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp Cell Res. 1995 ;217 (1) :57 -
40. Maione B, Lavitrano M, Spadafora C, Kiessling AA. Sperm-mediated gene transfer in mice. Mol Reprod Dev. 1998 ;50 (4) :406 -409
41. Lavitrano M, Stoppacciaro A, Bacci ML. et al. Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer. Transplant Proc. 1999 ;31 (1-2) :972 -974
42. Lazzereschi D, Forni M, Cappello F. et al. Efficiency of transgenesis using sperm-mediated gene transfer: Generation of hDAF transgenic pigs. Transplant Proc. 2000 ;32 (5) :892 -894
43. Zani M, Lavitrano M, French D. et al. The mechanism of binding of exogenous DNA to sperm cells - factors controlling the DNA uptake. Exp Cell Res. 1995 ;217 (1) :57 -64
44. Lavitrano M, French D, Zani M. et al. The interaction between exogenous DNA and sperm cells. Mol Reprod Dev. 1992 ;31 (3) :161 -169
45. Lavitrano M, Maione B, Forte E. et al. The interaction of sperm cells with exogenous DNA: A role of CD4 and major histocompatibility complex class II molecules. Exp Cell Res. 1997 ;233 (1) :56 -62
46. Sotolongo B, Lino E, Ward WS. Ability of hamster spermatozoa to digest their own DNA. Biol Reprod. 2003 ;69 (6) :2029 -2035
47. Spadafora C. Sperm cells and foreign DNA: a controversial relation. Bioessays. 1998 ;20 (11) :955 -964
48. Szczygiel MA, Moisyadi S, Ward WS. Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biol Reprod. 2003 ;68 (5) :1903 -1910
49. Francolini M, Lavitrano M, Lamia CL. et al. Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells. Mol Reprod Dev. 1993 ;34 (2) :133 -139
50. Camaioni A, Russo MA, Odorisio T. et al. Uptake of exogenous DNA by mammalian spermatozoa - specific localization of DNA on sperm heads. J Reprod Fertil. 1992 ;96 (1) :203 -212
51. Szczygiel MA, Moisyadi S, Ward WS. Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biol Reprod. 2003 ;68 (5) :1903 -1910
52. Perry ACF, Wakayama T, Kishikawa H. et al. Mammalian transgenesis by intracytoplasmic sperm injection. Science. 1999 ;284 (5417) :1180 -1183
53. Chan AWS, Luetjens CM, Dominko T. et al. Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live Rhesus monkey births. Mol Hum Reprod. 2000 ;6 (1) :26 -33
54. Chan AWS, Luetjens CM, Dominko T. et al. TransgenICSI reviewed: Foreign DNA transmission by intracytoplasmic sperm injection in rhesus monkey. Mol Reprod Dev. 2000 ;56 (2) :325 -328
55. Lai LX, Sun QY, Wu GM. et al. Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes. Zygote. 2001 ;9 (4) :339 -346
56. Nagashima H, Fujimura T, Takahagi Y. et al. Development of efficient strategies for the production of genetically modified pigs. Theriogenology. 2003 ;59 (1) :95 -106
57. Joris H, Nagy Z, Van de Velde H. et al. Intracytoplasmic sperm injection: laboratory set-up and injection procedure. Hum Reprod. 1998 ;13 :76 -86
58. Van Steirteghem A, Nagy P, Joris H. et al. Results of intracytoplasmic sperm injection with ejaculated, fresh and frozen-thawed epididymal and testicular spermatozoa. Hum Reprod. 1998 ;13 :134 -142
59. Mansour R. Intracytoplasmic sperm injection: a state of the art technique. Hum Reprod. 1998 ;4 (1) :43 -56
60. Huguet E, Esponda P. Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa. Mol Reprod Dev. 1998 ;51 (1) :42 -52
61. Huguet E, Esponda P. Generation of genetically modified mice by spermatozoa transfection in vivo: preliminary results. Mol Reprod Dev. 2000 ;56 (2) :243 -247
62. Sato M, Ishikawa A, Kimura M. Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa. Mol Reprod Dev. 2002 ;61 (1) :49 -56
63. Sato M, Gotoh K, Kimura M. Sperm-mediated gene transfer by direct injection of foreign DNA into mouse testis. Transgenics. 1999 ;2 (4) :357 -369
64. Sato M, Yabuki K, Watanabe T, Kimura M. Testis-mediated gene transfer (TMGT) in mice: Successful transmission of introduced DNA from F0 to F2 generations. Transgenics. 1999 ;3 (1) :11 -
65. Brinster RL. Germline stem cell transplantation and transgenesis. Science. 2002 ;296 (5576) :2174 -2176
66. Nagano M, Brinster CJ, Orwig KE. et al. Transgenic mice produced by retroviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A. 2001 ;98 (23) :13090 -13095
67. Orwig KE, Avarbock MR, Brinster RL. Retrovirus-mediated modification of male germline stem cells in rats. Biol Reprod. 2002 ;67 (3) :874 -879
68. Honaramooz A, Megee SO, Dobrinski I. Germ cell transplantation in pigs. Biol Reprod. 2002 ;66 (1) :21 -28
69. Honaramooz A, Behboodi E, Blash S. et al. Germ cell transplantation in goats. Mol Reprod Dev. 2003 ;64 (4) :422 -428
70. Ellis J, Bernstein A. Gene targeting with retroviral vectors - recombination by gene conversion into regions of nonhomology. Mol Cell Biol. 1989 ;9 (4) :1621 -1627
71. Smith K. Theoretical mechanisms in targeted and random integration of transgene DNA. Reprod Nutr Dev. 2001 ;41 (6) :465 -485
72. Colosimo A, Guida V, Palka G, Dallapiccola B. Extrachromosomal genes: a powerful tool in gene targeting approaches. Gene Ther. 2002 ;9 (11) :679 -682
73. Stoll SM, Calos MP. Extrachromosomal plasmid vectors for gene therapy. Curr Opin Mol Ther. 2002 ;4 (4) :299 -305
74. Khoo HW, Ang LH, Lim HB, Wong KY. Sperm Cells as Vectors for Introducing Foreign DNA into Zebrafish. Aquaculture. 1992 ;107 (1) :1 -19
75. Mannik A, Piirsoo M, Nordstrom K. et al. Effective generation of transgenic mice by Bovine papillomavirus type 1 based self-replicating plasmid that is maintained as extrachromosomal genetic element in three generations of animals. Plasmid. 2003 ;49 (3) :193 -204
76. Celebi C, Auvray P, Benvegnu T. et al. Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells. Mol Reprod Dev. 2002 ;62 (4) :477 -482
77. Mannik A, Runkorg K, Jaanson N. et al. Induction of the bovine papillomavirus origin "onion skin"-type DNA replication at high E1 protein concentrations in vivo. J Virol. 2002 ;76 (11) :5835 -5845
78. Lacey M, Alpert S, Hanahan D. Bovine papillomavirus genome elicits skin tumors in transgenic mice. Nature. 1986 ;322 (6080) :609 -612
79. De Jong G, Telenius A, Vanderbyl S. et al. Efficient in-vitro transfer of a 60-Mb mammalian artificial chromosome into murine and hamster cells using cationic lipids and dendrimers. Chromosome Res. 2001 ;9 (6) :475 -485
80. Grimes B, Cooke H. Engineering mammalian chromosomes. Hum Mol Genet. 1998 ;7 (10) :1635 -1640
81. Co DO, Borowski AH, Leung JD. et al. Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection. Chromosome Res. 2000 ;8 (3) :183 -191
82. Hadlaczky G. Satellite DNA-based artificial chromosomes for use in gene therapy. Curr Opin Mol Ther. 2001 ;3 (2) :125 -132
83. Choo KHA. Engineering human chromosomes for gene therapy studies. Trends in Molecular Medicine. 2001 ;7 (6) :235 -237
84. Grimes BR, Warburton PE, Farr CJ. Chromosome engineering: prospects for gene therapy. Gene Ther. 2002 ;9 (11) :713 -718
85. Lipps HJ, Jenke ACW, Nehlsen K. et al. Chromosome-based vectors for gene therapy. Gene Ther. 2003 ;304 :23 -33
86. Abbondanzo SJ, Gadi I, Stewart CL. Derivation of embryonic stem-cell lines. Guide to Techniques in Mouse Development. 1993 ;225 :803 -823
87. Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci U S A. 1997 ;94 (11) :5709 -5712
88. Robertson EJ. Embryo-derived stem cell lines. In: (ed.) Robertson EJ. Teratocarcinomas and embryonic stem cells, a practical approach. Oxford: IRL Press. 1987 :71-112
89. Torres M. The use of embryonic stem cells for the genetic manipulation of the mouse. Cellular and Molecular Procedures in Developmental Biology. 1998 ;36 :99 -114
90. Pirity M, Hadjantonakis AK, Nagy A. Embryonic stem cells, creating transgenic animals. Methods Cell Biol. 1998 ;57 :279 -293
91. Torres M. The use of embryonic stem cells for the genetic manipulation of the mouse. Curr Top Dev Biol. 1998 ;36 :99 -114
92. Metzger D, Feil R. Engineering the mouse genome by site-specific recombination. Curr Opin Biotechnol. 1999 ;10 (5) :470 -476
93. Muller U. Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev. 1999 ;82 (1-2) :3 -21
94. Prelle K, Vassiliev IM, Vassilieva SG. et al. Establishment of pluripotent cell lines from vertebrate species - Present status and future prospects. Cells Tissues Organs. 1999 ;165 (3-4) :220 -236
95. Wheeler MB, Walters EM, Clark SG. Transgenic animals in biomedicine and agriculture: outlook for the future. Anim Reprod Sci. 2003 ;79 (3-4) :265 -289
96. Thomson JA, Itskovitz-Eldor J, Shapiro SS. et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998 ;282 (5391) :1145 -1147
97. Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod. 2004 ;70 (3) :837 -845
98. Conley BJ, Trounson AO, Mollard R. Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives. Fetal Diagn Ther. 2004 ;19 (3) :218 -223
99. Park JH, Kim SJ, Oh EJ. et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod. 2003 ;69 (6) :2007 -2014
100. Reubinoff BE, Pera MF, Fong CY. et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000 ;18 (4) :399 -404
101. Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol. 2003 ;21 (3) :319 -321
102. Campbell KHS, McWhir J, Ritchie WA, Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line. Nature. 1996 :64-65
103. Schnieke AE, Kind AJ, Ritchie WA. et al. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science. 1997 ;278 (5346) :2130 -2133
104. Wilmut I. Viable offspring derived from fetal and adult mammalian cells. Societal, medical and ethical implications of cloning. London: Office for Official Publications of the European Communities. 1997 3-8
105. Tsunoda Y, Kato Y. The recent progress on nuclear transfer in mammals. Zoolog Sci. 2000 ;17 (9) :1177 -1184
106. Wolf E, Schernthaner W, Zakhartchenko V. et al. Transgenic technology in farm animals - progress and perspectives. Exp Physiol. 2000 ;85 (6) :615 -625
107. Hwang WS, Ryu YJ, Park JH. et al. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science. 2004 ;303 (5664) :1669 -1674
108. Clark AJ, Burl S, Denning C, Dickinson P. Gene targeting in livestock: a preview. Transgenic Res. 2000 ;9 (4-5) :263 -275
109. McCreath KJ, Howcroft J, Campbell KHS. et al. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature. 2000 ;405 (6790) :1066 -1069
110. Lai LX, Kolber-Simonds D, Park KW. et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer coning. Science. 2002 ;295 (5557) :1089 -1092
111. Smith LC, Bordignon V, Babkine M. et al. Benefits and problems with cloning animals. Can Vet J. 2000 ;41 (12) :919 -924
112. Renard JP, Zhou Q, LeBourhis D. et al. Nuclear transfer technologies: Between successes and doubts. Theriogenology. 2002 ;57 (1) :203 -222
113. Kono T. Influence of epigenetic changes during oocyte growth on nuclear reprogramming after nuclear transfer. Reprod Fertil Dev. 1998 ;10 (7-8) :593 -598
114. Rideout WM, Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome. Science. 2001 ;293 (5532) :1093 -1098
115. Wakayama T, Yanagimachi R. Mouse cloning with nucleus donor cells of different age and type. Mol Reprod Dev. 2001 ;58 (4) :376 -383
116. Riele HT, Maandag ER, Berns A. Highly efficient gene targeting in embryonic stem-cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci U S A. 1992 ;89 (11) :5128 -5132
117. Vandeursen J, Wieringa B. Targeting of the creatine kinase-M gene in embryonic stem-cells using isogenic and nonisogenic vectors. Nucleic Acids Res. 1992 ;20 (15) :3815 -3820
118. Hirata R, Chamberlain J, Dong R, Russell DW. Targeted transgene insertion into human chromosomes by adeno- associated virus vectors. Nat Biotechnol. 2002 ;20 (7) :735 -738
119. Porteus MH, Cathomen T, Weitzman MD, Baltimore D. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol Cell Biol. 2003 ;23 (10) :3558 -3565
120. Miller DG, Petek LM, Russell DW. Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol Cell Biol. 2003 ;23 (10) :3550 -3557
121. Chamberlain JR, Schwarze U, Wang PR. et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science. 2004 ;303 (5661) :1198 -1201
Kevin Smith lectures in genetics and physiology in the School of Contemporary Sciences, University of Abertay, Dundee, United Kingdom. His research interests include transgenesis, gene therapy and bioethics. He is an experienced review article writer and has published several excellent reviews in the fields of transgenesis, gene therapy and bioethics.
K. R. Smith, School of Contemporary Sciences, University of Abertay Dundee, Baxter Building, Dundee, DD1 1HG, United Kingdom. Phone: +44 (0) 01382 308664 Email: k.smithac.uk
Gene Transfer Methods