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Nonhuman primate transgenesis: progress and prospects
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Nonhuman primate transgenesis: progress and prospects Michael J. Wolfgang and Thaddeus G. Golos The nonhuman primate is used extensively in biomedical research owing to its close similarities to human physiology and human disease pathophysiology. Recently, several groups have initiated efforts to genetically manipulate nonhuman primates to address complex questions concerning primate-specific development and physiological adaptation. Primates pose unique challenges to transgenesis and, although this field is still in its infancy, the potential for obtaining new insights into primate physiology and gene function is unprecedented. This review focuses on the methods and potential applications of genetically altered nonhuman primates in biomedical research. Published online: 19 September 2002
Michael J. Wolfgang Yale University School of Medicine, Dept of Pathology, New Haven, CT 06520, USA. Thaddeus G. Golos* Wisconsin Regional Primate Research Center and the Dept. of Obstetrics & Gynecology, University of Wisconsin Medical School, Madison WI 53715, USA. *e-mail: [email protected]
The advent of gene targeting and transgenesis has fostered profound advances in biomedical research over the past several decades. Over-expression or ablation of specific genes, coupled with approaches for tissue- and cell-specific modification, has allowed great strides to be made in the understanding of the genetic determinants of development and the genetic components of disease. Model organisms such as Drosophila, Caenorhabditis elegans and the laboratory mouse, have provided most of this information because of their relative ease of manipulation, and the wealth of available genetic information. However, the development and physiology of even the mouse is sometimes disparate from humans and new models must be developed to address questions unique to primate physiology. Nonhuman primates have a long history of use in biomedical research owing to their close similarities to humans in embryonic development, reproduction and overall physiology. However, the opportunity to realize genetic modification of primates has awaited the commitment of animal resources, advances in nonhuman primate assisted reproductive technologies and gene transfer technologies. The use of nonhuman primates in in vivo molecular genetic studies has become feasible because of timely recent developments in all of these areas. The development of assisted reproductive technologies, such as in vitro fertilization, embryo culture and embryo transfer, has provided efficient means of establishing pregnancies from in vitro manipulated embryos [1–3]. Methods for efficient gene transfer arising from advances in the gene therapy arena could significantly enhance the production of genetically manipulated nonhuman primates by overcoming the inherent inefficiency of pronuclear injection encountered in most species other than the mouse [4]. http://tibtech.trends.com
Here we review the current standing of nonhuman primate transgenesis and describe promising embryonic gene transfer strategies and selected areas of biomedical research with the nonhuman primate model. Facilitating embryonic gene transfer
Whereas it is a relatively simple matter to obtain several hundred mouse oocytes to initiate a transgenesis project, nonhuman primate oocytes must be obtained from animals through laborious ovarian stimulation and oocyte retrieval protocols similar to those used in human in vitro fertilization (IVF) clinics. Owing to the relatively scarce resources available to primate embryologists, alternative gene transfer methods to pronuclear injection of DNA have been sought to facilitate more efficient transgenesis. A vector well suited for primate gene transfer should fulfill several criteria for successful transgenesis (Box 1). These criteria also need to be addressed by researchers in the gene therapy field; thus primate embryologists have exploited advances in gene therapy vector design. Episomal vectors
Transgene delivery by an episomal vector has several advantages over integrating vectors. First, because the transgene is not inserted into the host chromosome it is not affected by the local transcriptional environment at the integration site, such as the presence of transcriptional enhancers or repressors, the acetylation state of chromatin-associated histones, or the methylation of host DNA (or the transgene itself). A relatively consistent transgene copy number provided by episomal vectors should provide more uniform transgene expression, and improve homogeneity among groups and within animals, or embryos in this context. Given that integrating vectors, such as retroviruses, can activate DNA repair machinery [5], they could have unpredictable effects on early gene expression. In addition, insertional mutagenesis remains a concern with respect to integrating vectors, but less so with episomal vectors. There are several widely used episomal vectors; one example is based on adenovirus. Adenoviruses contain a double-stranded DNA genome, are nonintegrating and have been developed as vectors for gene therapy because of their highly efficient gene transfer to many cells and tissues, both in vitro and in vivo. The main limitation of this vector in somatic gene therapy is its inherent immunogenicity.
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Box 1. Criteria for successful gene transfer vectors in nonhuman primate embryonic transgenesis • Owing to the limiting number of oocytes, the vector must introduce the transgene into the zygote or embryo at high efficiency. • The transgene must be stable in transduced cells and faithfully segregate into all daughter cells. • The transgene must maintain its expression and be robust against epigenetic silencing. For many applications, a physiologically appropriate pattern will be desirable.
However, this is not a concern for embryologists because the fetus does not acquire adaptive immunity until much later in development. Mammalian embryos from several species can be transduced by adenovirus after perivitelline microinjection [6,7] or after removal of the zona pellucida [8]. An additional limitation of adenoviral vectors in transgenesis is instability as an episome. Furthermore, the use of the complex adenovirus genome could alter gene expression in the embryo. Embryos and embryonic stem cells express a protein that can substitute for the adenoviral transcription factor E1a [9–11], thus adenovirus transduction could potentially interfere with normal patterns of embryonic gene expression. Another episomal vector available for use in primate cells is based on components of the Epstein-Barr virus (EBV). EBV is a double-stranded herpesvirus that preferentially infects human and nonhuman primate B lymphocytes and can exist episomally as a latent infection. Investigators have exploited the latent infectious machinery of EBV to derive a novel vector system [12]. Vectors are designed to constitutively express Epstein Barr Deletion Constitutive promoter
-R-U5-Internal promoter-transgene-U3-R-U5 -Gag/Pol
-Envelope protein
-Rev HIV only
• VSV-G • Gibbon Ape-Leukemia Virus • Filovirus
Envelope protein 293T Cells
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Fig 1. Production of pseudotyped retroviral vectors. Replication-incompetent retroviruses can be produced by simple transfection of plasmids that carry the essential genes necessary for production of an infectious virion [30]. The viral genome is divided into nonoverlapping segments amongst several plasmids; constitutive promoters independent of viral regulatory elements direct transcription of the genes necessary for virion production and assembly. Envelope proteins expressed by another plasmid can be used to modify the tropism of the virus. These steps lower the risk of creating replicationcompetent virus via recombination of plasmids in the packaging cells. Additionally, retroviruses can contain a 3’ UTR deletion to make the vector self-inactivating [30–32]. Thus, the virions generated by the packaging cell are incapable of further replication in their target cell.
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Nuclear Antigen-1, which then associates the latent origin of replication (oriP ), located in the transgene vector, with nuclear chromatin. This association allows the segregation of vector into daughter cells because the vector is replicated along with chromosomal DNA in each cell cycle [12–14]. This system has not been tested extensively in other species because EBV does not maintain these associations in rodent cells [12]. It has, however, recently been evaluated in rhesus monkey zygotes by direct pronuclear injection [15] and might be useful for directing embryonic transgene expression (50% of injected embryos expressed the transgene). The attractiveness of this approach includes the simplicity of plasmid microinjection, without attendant issues of producing high titer vector tropic for zygotes or embryos. However, uncertainties of the maintenance of the vector through fetal development and in resulting offspring would seem to limit its applicability for promoting primate transgenesis. Integrating vectors
Vectors based on integrating retroviruses have been explored for embryonic gene transfer for nearly two decades [16] and are widely used for nonembryonic targets. Retroviruses are single-stranded positive RNA viruses that contain a diploid genome, although only one copy is integrated into the host cell per virion. The genome is packaged into a glycoprotein capsid and surrounded by a lipid bilayer derived from the host cell’s plasma membrane as the virion buds off from the host cell. This outer membrane contains envelope glycoproteins encoded by the viral genome that determine the host range of the virus. Retroviruses can be pseudotyped (i.e., they can be produced by packaging cell lines to express envelope proteins of other viruses), thus modifying the host range of the recombinant virion (Fig. 1). Several envelope proteins have been used to pseudotype retroviruses, including envelope glycoproteins from gibbon ape leukemia virus [17], vesicular stomatitis virus [18,19] and the Ebola filovirus [20]. Moloney murine leukemia virus (MMLV) is a simple retrovirus that can integrate its genetic material into the genome of actively dividing cells or into cells with a disrupted nuclear membrane. However, MMLV is not transported through an intact nuclear membrane. Transgenic cattle were produced by injecting MMLV-based vector into mature metaphase II oocytes that do not contain intact nuclear membranes, enabling integration of the transgene [19]. However, the offspring from these experiments did not apparently produce any transgene protein. Transgenic rhesus monkeys were recently generated in a similar way by Chan et al. using an MMLV vector pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), and containing a green fluorescent protein (GFP) transgene under the control of the elongation factor-1α or cytomegalovirus immediate early promoters [21]. The transduced
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Embryo flushing
In vitro fertilization and development
Injection of vector into blastocoel Transcervical transfer
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Fig 2. Schematic diagram of embryonic gene transfer in rhesus monkeys. Rhesus monkey blastocysts were obtained through in vitro fertilization and development or were recovered by nonsurgical flushing techniques. Virus was injected into blastocysts and the transduced blastocysts were transferred into recipients via nonsurgical transcervical embryo transfer [27,67]. Transgenic tissue, embryos or offspring can then be used in gene regulation and function studies.
oocytes were then fertilized by intracytoplasmic sperm injection and transferred to recipient females. Live offspring as well as several nonviable pregnancies resulted from these females, which is a common outcome from rhesus monkey assisted reproductive procedures. Some stillborn fetuses carried vector DNA and were reported to express the GFP transgene, and of the surviving infants, one carried the transgene in all cells and tissues examined. However, expression of GFP was not detected in this animal [21]. Although lack of expression could be owing to an inhospitable integration site, it is also possible that the MMLV vector was epigenetically silenced. It is known that MMLV vectors can be efficiently silenced even when they contain an internal promoter in multipotent cells, such as embryonic blastomeres [18], hematopoietic stem cells, embryonic stem cells [11] and embryonic carcinoma cells [22]. Previous studies in cattle have suggested similar transgene silencing [18,19]. Regardless, this work showed the feasibility of obtaining offspring with altered genotypes by viral vector gene transfer to primate oocytes. Other integrating vectors might hold promise for primate embryonic gene transfer that could possibly circumvent silencing issues associated with simple retroviruses. Lentiviruses (such as SIV, HIV and HTLV) are complex retroviruses that are able to transduce nondividing cells through transport and integration mechanisms that can proceed in the presence of the nuclear membrane [23]. These viruses naturally infect nondividing cells of the hematopoietic lineage and vectors derived from lentivirus are active in mouse embryonic stem cells [24] and hematopoietic stem cells [23,25], and their undifferentiated and http://tibtech.trends.com
Fig 3. Cultured primary trophoblasts from an enzymaticaly dispersed placenta of a rhesus monkey infant that was transduced with a lentiviral vector at the blastocyst stage. Left: phase contrast images; Right: epifluorescence images. The scale bar represents 50 µm.
differentiated derivatives. They are attractive candidates for embryonic gene transfer because these studies indicate that they are less sensitive to transgene silencing. An HIV-based vector expressing GFP [26] was used in our laboratory to transduce rhesus monkey preimplantation embryos (Fig. 2). For this study, the vector was injected into the blastocoel cavity of in vitro-produced blastocysts and the embryos were nonsurgically introduced into recipient females. At term, the placentas were evaluated and widespread placental expression of the transgene mRNA and protein was confirmed [27]. The protein was detected throughout the chorion of the placenta and expression was detected by western blot, direct fluorescence in cultured trophoblasts (Fig. 3), immunohistochemistry of placental tissues and by the production of maternal antibodies specific for the transgene. All offspring produced from this technique to date demonstrated robust placental transgene expression. However, these offspring have not yet demonstrated transgene expression or integration into the limited number of somatic tissues examined thus far. Blastocoel injections could have targeted the trophectoderm and hypoblast, and it is likely that introduction of vector at earlier stages of development will result in transgene expression in fetal as well as extraembryonic tissues. Interestingly, one of the offspring exhibited transgene expression in the umbilical cord at term. Given that the cord is derived from epiblast, it is possible that this animal will be shown to be chimeric on more extensive evaluation. The use of lentiviral vectors for gene transfer and transgenesis has now also been shown to be effective with rodent models [28,29]. Several laboratories have shown that mice can be produced
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at a relatively high efficiency and transgene expression can be seen robustly throughout the offspring. There seems to be little question that fully transgenic primates can be reliably achieved, given sufficient resources and optimization of the timing of vector administration, embryo transfer methods and choice of promoter and transgene. There have been several improvements to the lentiviral vectors as originally described [23]. Most of the accessory HIV genes implicated in disease pathogenicity have been deleted and the vector has been further divided into separate plasmids to lower the risk of creating replication competent virus [30]. The vectors can be rendered self inactivating through a deletion in the 3′ regulatory region: after reverse transcription in the host cell, the deletion is added to the 5′ end of the proviral cDNA, thus transferring it to a crucial region of the vector promoter and making the vector itself transcriptionally inactive [31–33]. The addition of an intron in the transgene cassette [34–36] can influence the expression of the transgene. Most recently, the addition of other elements, such as the woodchuck hepatitis virus posttranscriptional regulatory element, has been demonstrated to increase transgene expression [26,37]. Further modifications, such as the addition of insulators [38], locus control regions [39], or scaffold attachment regions [40–42], might influence transgene expression by protecting the transgene from positional effects.
Future directions
Nonviral methods of transgene delivery
Embryonic and placental development
Other methods of transgene delivery could also be useful for primate transgenesis. Transgenic mice have been produced at a high efficiency by incubating membrane-disrupted sperm (by freeze thawing or detergent washing) with DNA and performing intracytoplasmic sperm injection [43,44]. This has worked well in mice and although it has not translated well in preliminary studies with monkeys [45] it might very well be a means of producing transgenic primates because good success with ICSI is readily obtained in rhesus monkeys [46,47]. Nuclear transfer techniques are being actively investigated in nonhuman primates to facilitate cloning of genetically important individuals and could potentially provide an avenue for the genetic modification of primates. Rhesus monkey offspring have been produced from the transfer of blastomere nuclei [48] but transfer of nuclei from somatic or embryonic stem (ES) cells has not been successful [49]. If somatic cell nuclear transfer is shown to be successful, this would be an excellent means of altering or selecting the genotypes of primate offspring as it has in other species [50,51]. Theoretically, the gene of interest could be flanked with loxP sites in nuclear donor cells and tissue-specific gene inactivation by embryonic or postnatal transduction with a vector expressing Cre recombinase [52] under the control of a cell-specific promoter could be accomplished.
Embryonic development in primates differs substantially from that in rodents, especially during the periimplantation period. Differences in the formation of basic postimplantation embryonic structures include morphogenesis and in some aspects the function of the placenta. Two areas in which nonhuman primates are uniquely suited include placental endocrinology and the placental contribution to maternal-fetal immune tolerance. Although the fetus expresses paternal antigens, it is not rejected as an allograft by the maternal immune system during mammalian gestation. The placenta undoubtedly has a central role here, although the precise mechanisms by which this maternal tolerance of the fetal semiallograft is generated remain incompletely understood. The hemochorial placenta of the rhesus macaque has close homology with that of the human, including organization into chorionic villi, a syncytial trophoblast layer in direct contact with maternal blood and extravillous trophoblasts that invade the maternal endometrium and have a role in vascular reorganization in response to pregnancy [55]. Although the rodent placenta is also hemochorial, the expression and distribution of immune regulatory molecules at the maternal-fetal interface is quite different between primates and rodents. An area of particular interest in the primate placenta is the preferential expression of nonpolymorphic major
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An alternative to adult somatic cell transfers is to produce transgenic primates using stably transfected primate embryonic stem cells [53]. ES cell injection into tetraploid embryos has been used to produce mice that are fully derived from the ES cells because the tetraploid embryos (produced by fusion of two-cell embryos) give rise solely to extraembryonic tissue [54]. Although many elegant experiments can be envisioned using these approaches, it needs to be emphasized that even in species for which resources are not limiting, such as mice or pigs, these techniques are very inefficient and the feasibility of these approaches in primate embryology remains to be demonstrated.
The cost and time involved in developing and maintaining transgenic nonhuman primates and the relatively long generation interval (puberty is achieved at ~4 years of age) in rhesus monkeys precludes the use of these animals in every avenue of research. The scientific and ethical justification for the use of primates remains, to study systems that are specific to primates or that cannot be studied in other species. Therefore, there are several areas where we foresee a need for genetically modified primates to expand our knowledge of human physiology, development and disease. These include, but are not limited to, embryonic development and placentation, immunobiology, virology and neurobiology.
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histocompatibility complex (MHC) class I molecules: these include HLA-G [56,57] and HLA-E [58] in the human placenta, and Mamu-AG [59–61] and Mamu-E [62] in the rhesus placenta. The transgenic primate model provides a way to investigate directly the importance of the expression of selected MHC loci during pregnancy. Modulation of the expression of MHC class I genes in the placenta, or other immune modulators or cytokines, could aid in defining the placental dialogue with the maternal immune system in the establishment and success of pregnancy. Immunobiology and virology
Acknowledgments We thank R. Becker for assistance with preparation of the figures, S. Busch for assistance with the preparation of this manuscript, and we acknowledge I. Verma and R. Hawley for reagents for the studies conducted in our laboratory. Supported by NIH grants RR14040 and RR00167. This is publication number 41-017 of the W.P.R.C.
Because of the close evolutionary relationship between humans and nonhuman primates, they are an important model for studying the pathophysiology of infectious disease. One of the most important uses of the nonhuman primate is as a model in efforts to understand, control and prevent human infection by HIV. A crucial area in current research with the nonhuman primate model is in understanding how the cytotoxic lymphocyte (CTL)-restricted immune response can be manipulated to prevent infection or control disease progression [63–65]. Because monkeys are an outbred population, the numbers of animals with specific MHC alleles or haplotypes can be limiting – a circumstance also complicated by the long intergenerational time. The use of transgenic technology could help to provide animals expressing selected MHC alleles for a precise determination of the role of a specific MHC allele in the immune response to simian immunodeficiency virus infection. Neurobiology
Nonhuman primates have a crucial role in neurobiological research because of the close similarities with humans in neuroanatomy and physiology. Because of the opportunities for genetic manipulation, transgenic and knock-out mice are
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currently the primary models for studies of the roles of specific genes in normal development and physiology of the nervous system, however, these studies can be difficult to extrapolate to human brain function. Gene transfer technology might serve an important role in several areas. Cell-specific expression of marker genes such as GFP can allow studies of neuron origin and migration during fetal development. Marking of cells could allow investigators to readily identify and isolate cells of interest for single-cell electrophysiological or molecular analysis, or for isolation and clonal analysis of putative neural stem cells. Gene transfer technology has become increasingly important because of the pressing need to develop cellular and molecular therapies for neurodegenerative diseases, accelerated by the advent of therapeutic stem cell opportunities. Summary and outlook
Advances in molecular medicine and gene therapy vector technology have allowed investigators to approach primate transgenesis as a new tool for understanding basic human and nonhuman primate biology, as well as to develop new approaches for understanding and treating human disease. Practical and ethical limitations will help investigators focus on those crucial areas for which primates are unique models, and have traditionally been important partners in biomedical research. Concerns about ‘appropriate’ experiments to conduct with primates with regards to genetic modification are an important area to address in dialogue with the public [66]. It will be important to insure that the use of genetic technology does not negatively impact on the use of primates in biomedical research. In particular, the intense public debate on human cloning, embryonic stem cells, genetic selection and genetic modification counsel biomedical researchers to focus clearly on the advances in human health and medicine offered by use of the nonhuman primate model.
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Congratulations! Congratulations to G.N.M. Ferreira who won first prize in the Trends in Biotechnology Poster Award at the ESBES-4 symposium (held in Delft, August 2002). His poster entitled ‘Development of DNA biochips for genomic analysis’ described the development of a new generation of biochips aimed at the integration of detection systems in ‘user friendly’ devices. Second prize went to M.A. Hoeben for his poster entitled ‘Separation of bioparticles by interfacial partitioning’ and the winner of the third prize was P.A. Bird, whose poster was entitled ‘Monitoring and control: the Baeyer-Villiger monooxygenase catalysed synthesis of an optically pure lactone’.
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Nonhuman primate transgenesis: progress and prospects Michael J. Wolfgang and Thaddeus G. Golos The nonhuman primate is used extensively in biomedical research owing to its close similarities to human physiology and human disease pathophysiology. Recently, several groups have initiated efforts to genetically manipulate nonhuman primates to address complex questions concerning primate-specific development and physiological adaptation. Primates pose unique challenges to transgenesis and, although this field is still in its infancy, the potential for obtaining new insights into primate physiology and gene function is unprecedented. This review focuses on the methods and potential applications of genetically altered nonhuman primates in biomedical research. Published online: 19 September 2002
Michael J. Wolfgang Yale University School of Medicine, Dept of Pathology, New Haven, CT 06520, USA. Thaddeus G. Golos* Wisconsin Regional Primate Research Center and the Dept. of Obstetrics & Gynecology, University of Wisconsin Medical School, Madison WI 53715, USA. *e-mail: [email protected]
The advent of gene targeting and transgenesis has fostered profound advances in biomedical research over the past several decades. Over-expression or ablation of specific genes, coupled with approaches for tissue- and cell-specific modification, has allowed great strides to be made in the understanding of the genetic determinants of development and the genetic components of disease. Model organisms such as Drosophila, Caenorhabditis elegans and the laboratory mouse, have provided most of this information because of their relative ease of manipulation, and the wealth of available genetic information. However, the development and physiology of even the mouse is sometimes disparate from humans and new models must be developed to address questions unique to primate physiology. Nonhuman primates have a long history of use in biomedical research owing to their close similarities to humans in embryonic development, reproduction and overall physiology. However, the opportunity to realize genetic modification of primates has awaited the commitment of animal resources, advances in nonhuman primate assisted reproductive technologies and gene transfer technologies. The use of nonhuman primates in in vivo molecular genetic studies has become feasible because of timely recent developments in all of these areas. The development of assisted reproductive technologies, such as in vitro fertilization, embryo culture and embryo transfer, has provided efficient means of establishing pregnancies from in vitro manipulated embryos [1–3]. Methods for efficient gene transfer arising from advances in the gene therapy arena could significantly enhance the production of genetically manipulated nonhuman primates by overcoming the inherent inefficiency of pronuclear injection encountered in most species other than the mouse [4]. http://tibtech.trends.com
Here we review the current standing of nonhuman primate transgenesis and describe promising embryonic gene transfer strategies and selected areas of biomedical research with the nonhuman primate model. Facilitating embryonic gene transfer
Whereas it is a relatively simple matter to obtain several hundred mouse oocytes to initiate a transgenesis project, nonhuman primate oocytes must be obtained from animals through laborious ovarian stimulation and oocyte retrieval protocols similar to those used in human in vitro fertilization (IVF) clinics. Owing to the relatively scarce resources available to primate embryologists, alternative gene transfer methods to pronuclear injection of DNA have been sought to facilitate more efficient transgenesis. A vector well suited for primate gene transfer should fulfill several criteria for successful transgenesis (Box 1). These criteria also need to be addressed by researchers in the gene therapy field; thus primate embryologists have exploited advances in gene therapy vector design. Episomal vectors
Transgene delivery by an episomal vector has several advantages over integrating vectors. First, because the transgene is not inserted into the host chromosome it is not affected by the local transcriptional environment at the integration site, such as the presence of transcriptional enhancers or repressors, the acetylation state of chromatin-associated histones, or the methylation of host DNA (or the transgene itself). A relatively consistent transgene copy number provided by episomal vectors should provide more uniform transgene expression, and improve homogeneity among groups and within animals, or embryos in this context. Given that integrating vectors, such as retroviruses, can activate DNA repair machinery [5], they could have unpredictable effects on early gene expression. In addition, insertional mutagenesis remains a concern with respect to integrating vectors, but less so with episomal vectors. There are several widely used episomal vectors; one example is based on adenovirus. Adenoviruses contain a double-stranded DNA genome, are nonintegrating and have been developed as vectors for gene therapy because of their highly efficient gene transfer to many cells and tissues, both in vitro and in vivo. The main limitation of this vector in somatic gene therapy is its inherent immunogenicity.
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Box 1. Criteria for successful gene transfer vectors in nonhuman primate embryonic transgenesis • Owing to the limiting number of oocytes, the vector must introduce the transgene into the zygote or embryo at high efficiency. • The transgene must be stable in transduced cells and faithfully segregate into all daughter cells. • The transgene must maintain its expression and be robust against epigenetic silencing. For many applications, a physiologically appropriate pattern will be desirable.
However, this is not a concern for embryologists because the fetus does not acquire adaptive immunity until much later in development. Mammalian embryos from several species can be transduced by adenovirus after perivitelline microinjection [6,7] or after removal of the zona pellucida [8]. An additional limitation of adenoviral vectors in transgenesis is instability as an episome. Furthermore, the use of the complex adenovirus genome could alter gene expression in the embryo. Embryos and embryonic stem cells express a protein that can substitute for the adenoviral transcription factor E1a [9–11], thus adenovirus transduction could potentially interfere with normal patterns of embryonic gene expression. Another episomal vector available for use in primate cells is based on components of the Epstein-Barr virus (EBV). EBV is a double-stranded herpesvirus that preferentially infects human and nonhuman primate B lymphocytes and can exist episomally as a latent infection. Investigators have exploited the latent infectious machinery of EBV to derive a novel vector system [12]. Vectors are designed to constitutively express Epstein Barr Deletion Constitutive promoter
-R-U5-Internal promoter-transgene-U3-R-U5 -Gag/Pol
-Envelope protein
-Rev HIV only
• VSV-G • Gibbon Ape-Leukemia Virus • Filovirus
Envelope protein 293T Cells
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Fig 1. Production of pseudotyped retroviral vectors. Replication-incompetent retroviruses can be produced by simple transfection of plasmids that carry the essential genes necessary for production of an infectious virion [30]. The viral genome is divided into nonoverlapping segments amongst several plasmids; constitutive promoters independent of viral regulatory elements direct transcription of the genes necessary for virion production and assembly. Envelope proteins expressed by another plasmid can be used to modify the tropism of the virus. These steps lower the risk of creating replicationcompetent virus via recombination of plasmids in the packaging cells. Additionally, retroviruses can contain a 3’ UTR deletion to make the vector self-inactivating [30–32]. Thus, the virions generated by the packaging cell are incapable of further replication in their target cell.
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Nuclear Antigen-1, which then associates the latent origin of replication (oriP ), located in the transgene vector, with nuclear chromatin. This association allows the segregation of vector into daughter cells because the vector is replicated along with chromosomal DNA in each cell cycle [12–14]. This system has not been tested extensively in other species because EBV does not maintain these associations in rodent cells [12]. It has, however, recently been evaluated in rhesus monkey zygotes by direct pronuclear injection [15] and might be useful for directing embryonic transgene expression (50% of injected embryos expressed the transgene). The attractiveness of this approach includes the simplicity of plasmid microinjection, without attendant issues of producing high titer vector tropic for zygotes or embryos. However, uncertainties of the maintenance of the vector through fetal development and in resulting offspring would seem to limit its applicability for promoting primate transgenesis. Integrating vectors
Vectors based on integrating retroviruses have been explored for embryonic gene transfer for nearly two decades [16] and are widely used for nonembryonic targets. Retroviruses are single-stranded positive RNA viruses that contain a diploid genome, although only one copy is integrated into the host cell per virion. The genome is packaged into a glycoprotein capsid and surrounded by a lipid bilayer derived from the host cell’s plasma membrane as the virion buds off from the host cell. This outer membrane contains envelope glycoproteins encoded by the viral genome that determine the host range of the virus. Retroviruses can be pseudotyped (i.e., they can be produced by packaging cell lines to express envelope proteins of other viruses), thus modifying the host range of the recombinant virion (Fig. 1). Several envelope proteins have been used to pseudotype retroviruses, including envelope glycoproteins from gibbon ape leukemia virus [17], vesicular stomatitis virus [18,19] and the Ebola filovirus [20]. Moloney murine leukemia virus (MMLV) is a simple retrovirus that can integrate its genetic material into the genome of actively dividing cells or into cells with a disrupted nuclear membrane. However, MMLV is not transported through an intact nuclear membrane. Transgenic cattle were produced by injecting MMLV-based vector into mature metaphase II oocytes that do not contain intact nuclear membranes, enabling integration of the transgene [19]. However, the offspring from these experiments did not apparently produce any transgene protein. Transgenic rhesus monkeys were recently generated in a similar way by Chan et al. using an MMLV vector pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G), and containing a green fluorescent protein (GFP) transgene under the control of the elongation factor-1α or cytomegalovirus immediate early promoters [21]. The transduced
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Embryo flushing
In vitro fertilization and development
Injection of vector into blastocoel Transcervical transfer
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Fig 2. Schematic diagram of embryonic gene transfer in rhesus monkeys. Rhesus monkey blastocysts were obtained through in vitro fertilization and development or were recovered by nonsurgical flushing techniques. Virus was injected into blastocysts and the transduced blastocysts were transferred into recipients via nonsurgical transcervical embryo transfer [27,67]. Transgenic tissue, embryos or offspring can then be used in gene regulation and function studies.
oocytes were then fertilized by intracytoplasmic sperm injection and transferred to recipient females. Live offspring as well as several nonviable pregnancies resulted from these females, which is a common outcome from rhesus monkey assisted reproductive procedures. Some stillborn fetuses carried vector DNA and were reported to express the GFP transgene, and of the surviving infants, one carried the transgene in all cells and tissues examined. However, expression of GFP was not detected in this animal [21]. Although lack of expression could be owing to an inhospitable integration site, it is also possible that the MMLV vector was epigenetically silenced. It is known that MMLV vectors can be efficiently silenced even when they contain an internal promoter in multipotent cells, such as embryonic blastomeres [18], hematopoietic stem cells, embryonic stem cells [11] and embryonic carcinoma cells [22]. Previous studies in cattle have suggested similar transgene silencing [18,19]. Regardless, this work showed the feasibility of obtaining offspring with altered genotypes by viral vector gene transfer to primate oocytes. Other integrating vectors might hold promise for primate embryonic gene transfer that could possibly circumvent silencing issues associated with simple retroviruses. Lentiviruses (such as SIV, HIV and HTLV) are complex retroviruses that are able to transduce nondividing cells through transport and integration mechanisms that can proceed in the presence of the nuclear membrane [23]. These viruses naturally infect nondividing cells of the hematopoietic lineage and vectors derived from lentivirus are active in mouse embryonic stem cells [24] and hematopoietic stem cells [23,25], and their undifferentiated and http://tibtech.trends.com
Fig 3. Cultured primary trophoblasts from an enzymaticaly dispersed placenta of a rhesus monkey infant that was transduced with a lentiviral vector at the blastocyst stage. Left: phase contrast images; Right: epifluorescence images. The scale bar represents 50 µm.
differentiated derivatives. They are attractive candidates for embryonic gene transfer because these studies indicate that they are less sensitive to transgene silencing. An HIV-based vector expressing GFP [26] was used in our laboratory to transduce rhesus monkey preimplantation embryos (Fig. 2). For this study, the vector was injected into the blastocoel cavity of in vitro-produced blastocysts and the embryos were nonsurgically introduced into recipient females. At term, the placentas were evaluated and widespread placental expression of the transgene mRNA and protein was confirmed [27]. The protein was detected throughout the chorion of the placenta and expression was detected by western blot, direct fluorescence in cultured trophoblasts (Fig. 3), immunohistochemistry of placental tissues and by the production of maternal antibodies specific for the transgene. All offspring produced from this technique to date demonstrated robust placental transgene expression. However, these offspring have not yet demonstrated transgene expression or integration into the limited number of somatic tissues examined thus far. Blastocoel injections could have targeted the trophectoderm and hypoblast, and it is likely that introduction of vector at earlier stages of development will result in transgene expression in fetal as well as extraembryonic tissues. Interestingly, one of the offspring exhibited transgene expression in the umbilical cord at term. Given that the cord is derived from epiblast, it is possible that this animal will be shown to be chimeric on more extensive evaluation. The use of lentiviral vectors for gene transfer and transgenesis has now also been shown to be effective with rodent models [28,29]. Several laboratories have shown that mice can be produced
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at a relatively high efficiency and transgene expression can be seen robustly throughout the offspring. There seems to be little question that fully transgenic primates can be reliably achieved, given sufficient resources and optimization of the timing of vector administration, embryo transfer methods and choice of promoter and transgene. There have been several improvements to the lentiviral vectors as originally described [23]. Most of the accessory HIV genes implicated in disease pathogenicity have been deleted and the vector has been further divided into separate plasmids to lower the risk of creating replication competent virus [30]. The vectors can be rendered self inactivating through a deletion in the 3′ regulatory region: after reverse transcription in the host cell, the deletion is added to the 5′ end of the proviral cDNA, thus transferring it to a crucial region of the vector promoter and making the vector itself transcriptionally inactive [31–33]. The addition of an intron in the transgene cassette [34–36] can influence the expression of the transgene. Most recently, the addition of other elements, such as the woodchuck hepatitis virus posttranscriptional regulatory element, has been demonstrated to increase transgene expression [26,37]. Further modifications, such as the addition of insulators [38], locus control regions [39], or scaffold attachment regions [40–42], might influence transgene expression by protecting the transgene from positional effects.
Future directions
Nonviral methods of transgene delivery
Embryonic and placental development
Other methods of transgene delivery could also be useful for primate transgenesis. Transgenic mice have been produced at a high efficiency by incubating membrane-disrupted sperm (by freeze thawing or detergent washing) with DNA and performing intracytoplasmic sperm injection [43,44]. This has worked well in mice and although it has not translated well in preliminary studies with monkeys [45] it might very well be a means of producing transgenic primates because good success with ICSI is readily obtained in rhesus monkeys [46,47]. Nuclear transfer techniques are being actively investigated in nonhuman primates to facilitate cloning of genetically important individuals and could potentially provide an avenue for the genetic modification of primates. Rhesus monkey offspring have been produced from the transfer of blastomere nuclei [48] but transfer of nuclei from somatic or embryonic stem (ES) cells has not been successful [49]. If somatic cell nuclear transfer is shown to be successful, this would be an excellent means of altering or selecting the genotypes of primate offspring as it has in other species [50,51]. Theoretically, the gene of interest could be flanked with loxP sites in nuclear donor cells and tissue-specific gene inactivation by embryonic or postnatal transduction with a vector expressing Cre recombinase [52] under the control of a cell-specific promoter could be accomplished.
Embryonic development in primates differs substantially from that in rodents, especially during the periimplantation period. Differences in the formation of basic postimplantation embryonic structures include morphogenesis and in some aspects the function of the placenta. Two areas in which nonhuman primates are uniquely suited include placental endocrinology and the placental contribution to maternal-fetal immune tolerance. Although the fetus expresses paternal antigens, it is not rejected as an allograft by the maternal immune system during mammalian gestation. The placenta undoubtedly has a central role here, although the precise mechanisms by which this maternal tolerance of the fetal semiallograft is generated remain incompletely understood. The hemochorial placenta of the rhesus macaque has close homology with that of the human, including organization into chorionic villi, a syncytial trophoblast layer in direct contact with maternal blood and extravillous trophoblasts that invade the maternal endometrium and have a role in vascular reorganization in response to pregnancy [55]. Although the rodent placenta is also hemochorial, the expression and distribution of immune regulatory molecules at the maternal-fetal interface is quite different between primates and rodents. An area of particular interest in the primate placenta is the preferential expression of nonpolymorphic major
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An alternative to adult somatic cell transfers is to produce transgenic primates using stably transfected primate embryonic stem cells [53]. ES cell injection into tetraploid embryos has been used to produce mice that are fully derived from the ES cells because the tetraploid embryos (produced by fusion of two-cell embryos) give rise solely to extraembryonic tissue [54]. Although many elegant experiments can be envisioned using these approaches, it needs to be emphasized that even in species for which resources are not limiting, such as mice or pigs, these techniques are very inefficient and the feasibility of these approaches in primate embryology remains to be demonstrated.
The cost and time involved in developing and maintaining transgenic nonhuman primates and the relatively long generation interval (puberty is achieved at ~4 years of age) in rhesus monkeys precludes the use of these animals in every avenue of research. The scientific and ethical justification for the use of primates remains, to study systems that are specific to primates or that cannot be studied in other species. Therefore, there are several areas where we foresee a need for genetically modified primates to expand our knowledge of human physiology, development and disease. These include, but are not limited to, embryonic development and placentation, immunobiology, virology and neurobiology.
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histocompatibility complex (MHC) class I molecules: these include HLA-G [56,57] and HLA-E [58] in the human placenta, and Mamu-AG [59–61] and Mamu-E [62] in the rhesus placenta. The transgenic primate model provides a way to investigate directly the importance of the expression of selected MHC loci during pregnancy. Modulation of the expression of MHC class I genes in the placenta, or other immune modulators or cytokines, could aid in defining the placental dialogue with the maternal immune system in the establishment and success of pregnancy. Immunobiology and virology
Acknowledgments We thank R. Becker for assistance with preparation of the figures, S. Busch for assistance with the preparation of this manuscript, and we acknowledge I. Verma and R. Hawley for reagents for the studies conducted in our laboratory. Supported by NIH grants RR14040 and RR00167. This is publication number 41-017 of the W.P.R.C.
Because of the close evolutionary relationship between humans and nonhuman primates, they are an important model for studying the pathophysiology of infectious disease. One of the most important uses of the nonhuman primate is as a model in efforts to understand, control and prevent human infection by HIV. A crucial area in current research with the nonhuman primate model is in understanding how the cytotoxic lymphocyte (CTL)-restricted immune response can be manipulated to prevent infection or control disease progression [63–65]. Because monkeys are an outbred population, the numbers of animals with specific MHC alleles or haplotypes can be limiting – a circumstance also complicated by the long intergenerational time. The use of transgenic technology could help to provide animals expressing selected MHC alleles for a precise determination of the role of a specific MHC allele in the immune response to simian immunodeficiency virus infection. Neurobiology
Nonhuman primates have a crucial role in neurobiological research because of the close similarities with humans in neuroanatomy and physiology. Because of the opportunities for genetic manipulation, transgenic and knock-out mice are
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currently the primary models for studies of the roles of specific genes in normal development and physiology of the nervous system, however, these studies can be difficult to extrapolate to human brain function. Gene transfer technology might serve an important role in several areas. Cell-specific expression of marker genes such as GFP can allow studies of neuron origin and migration during fetal development. Marking of cells could allow investigators to readily identify and isolate cells of interest for single-cell electrophysiological or molecular analysis, or for isolation and clonal analysis of putative neural stem cells. Gene transfer technology has become increasingly important because of the pressing need to develop cellular and molecular therapies for neurodegenerative diseases, accelerated by the advent of therapeutic stem cell opportunities. Summary and outlook
Advances in molecular medicine and gene therapy vector technology have allowed investigators to approach primate transgenesis as a new tool for understanding basic human and nonhuman primate biology, as well as to develop new approaches for understanding and treating human disease. Practical and ethical limitations will help investigators focus on those crucial areas for which primates are unique models, and have traditionally been important partners in biomedical research. Concerns about ‘appropriate’ experiments to conduct with primates with regards to genetic modification are an important area to address in dialogue with the public [66]. It will be important to insure that the use of genetic technology does not negatively impact on the use of primates in biomedical research. In particular, the intense public debate on human cloning, embryonic stem cells, genetic selection and genetic modification counsel biomedical researchers to focus clearly on the advances in human health and medicine offered by use of the nonhuman primate model.
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Congratulations! Congratulations to G.N.M. Ferreira who won first prize in the Trends in Biotechnology Poster Award at the ESBES-4 symposium (held in Delft, August 2002). His poster entitled ‘Development of DNA biochips for genomic analysis’ described the development of a new generation of biochips aimed at the integration of detection systems in ‘user friendly’ devices. Second prize went to M.A. Hoeben for his poster entitled ‘Separation of bioparticles by interfacial partitioning’ and the winner of the third prize was P.A. Bird, whose poster was entitled ‘Monitoring and control: the Baeyer-Villiger monooxygenase catalysed synthesis of an optically pure lactone’.
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