Search

Monogenic disorders occur at a high frequency in human populations and are commonly inherited through the germline. Unfortunately, once the mutation has been transmitted to a child, only limited treatment options are available in most cases. However, means of correcting disease-causing nuclear and mitochondrial DNA mutations in gametes or preimplantation embryos have now been developed and are commonly referred to as germline gene therapy (GGT). We will discuss these novel strategies and provide a path forward for safe, high-efficiency GGT that may provide a promising new paradigm for preventing the passage of deleterious genes from parent to child. Germline gene therapy has the potential to prevent inheritance of monogenic disorders but must first be optimized for accuracy., Author(s): Don P. Wolf [sup.1] , Paul A. Mitalipov [sup.1] , Shoukhrat M. Mitalipov [sup.1] Author Affiliations: (1) Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, [...]

Abstract Background Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT) has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. Results A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. Conclusion The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

The species-specific ovarian stimulation protocols and semen collection techniques have to be formed for consideration of vervets as tools for basic and applied assisted reproductive technological (ARTs) research on primate gametes or embryos.

Embryonic stem cells (ESCs) can proliferate indefinitely, maintain an undifferentiated pluripotent state and differentiate into any cell type. Differentiation of ESCs into various specific cell-types may be able to cure or alleviate the symptoms of various degenerative diseases. Unresolved issues regarding maintaining function, possible apoptosis and tumor formation in vivo mean a prudent approach should be taken towards advancing ESCs into human clinical trials. Rhesus macaques provide the ideal model organism for testing the feasibility, efficacy and safety of ESC based therapies and significant numbers of primate ESC lines are now available. In this review, we will summarize progress in evaluating the genetic and epigenetic integrity of primate ESCs, examine their current use in pre-clinical trials and discuss the potential of producing ESC-derived cell populations that are genetically identical (isogenic) to the host by somatic cell nuclear transfer.

Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction-based amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.

The nonhuman primate is a highly relevant model for the study of human diseases, and currently there is a significant need for populations of animals with specific genotypes that can not be satisfied by the capture of animals from the wild or by conventional breeding. There is an even greater need for genetically identical animals in vaccine development or tissue transplantation research, where immune system function is under study. Efficient somatic cell nuclear transfer (SCNT) procedures could provide a source for genetically identical nonhuman primates for biomedical research. SCNT offers the possibility of cloning animals using cultured cells and potentially provides an alternative approach for the genetic modification of primates. The opportunity to introduce precise genetic modifications into cultured cells by gene targeting procedures, and then use these cells as nuclear donors in SCNT, has potential application in the production of loss-of-function monkey models of human diseases. We were initially successful in producing monkeys by NT using embryonic blastomeres as the source of donor nuclei and have repeated that success. However, when somatic cells are used as nuclear donor cells, the developmental potential of monkey SCNT embryos is limited, and somatic cell cloning has not yet been accomplished in primates. High rates of in vitro development to blastocysts, comparable with in vitro fertilization controls, and successful production of rhesus monkeys by NT from embryonic blastomeres suggests that basic cloning procedures, including enucleation, fusion, and activation, are consistent with the production of viable embryos. Although modifications or additional steps in SCNT are clearly warranted, the basic procedures will likely be similar to those extant for embryonic cell NT. In this chapter, we describe detailed protocols for rhesus macaque embryonic cell NT, including oocyte and embryo production, micromanipulation, and embryo transfer in nonhuman primates.