This R24 research resource renewal will produce transgene expressing primate offspring and sets of genetically identical offspring. Ten questions in three aims are posed.
Aim 1 : to produce at least ten cloned NHPs by overcoming unanticipated hurdles in cytoplasmic inheritance just discovered during primate cloning. Will pronuclear removal after fertilization and cloning succeed in overcoming defects caused by meiotic spindle removal and the limitations of artificial activation? Will double NT succeed in overcoming problems of meiotic spindle removal, artificial activation, and sperm centrosome absence? Will spindle collapse permit maternal chromosome extraction to support somatic cell nuclear transfer (SCNT) pre- and post-implantation development? Does cytoplasmic supplementation restore proteins discarded during enucleation? Aim 2: to produce at least eight sets of identical offspring through embryo splitting. Are primates derived by embryo splitting valid research models? Will androgenote or tetraploid embryos support primate pregnancies, without significant contribution to the offspring, as is performed routinely for generating mouse-embryonic stem cell (ESC) offspring? Aim 3: to produce at least 16 transgene-expressing primate offspring. Will lentivirus transgenesis, which promises dramatic improvements in efficiency, integration, expression, transmission, succeed in generating transgene-expressing primates? Can retroviral vectors be improved to generate transgene-expressing primates? Will transgenesis by intracytoplasmic sperm injection, ICSI, (transgenlCSI) succeed in generating transgene-expressing offspring with stable DNA integration? Will direct pronuclear injection prove more reliable for stable integration than transgenlCSl? Performing these applied primate studies will develop new research resources for producing and propagating invaluable biomedical models, as well as determining the feasibility of human embryonic stem cell (HESC) potentials. This investigation continues the resource development of invaluable, but not yet proven or perfected technologies, for reliably and routinely generating cloned and transgenic NHPs. By expanding the manners in which NHP offspring are propagated, the utility of this model for important research is greatly enhanced. SCNT may well find exceptional utility as a reliable, routine approach for generating essential specimens. The production of transgenic NHPs as the most clinically relevant models for human diseases might well be of vital biomedical importance. Furthermore, the promise of safe and effective gene therapy protocols cannot be fully realized until an appropriate system for investigation is found to fill the gap between transgenic mice and seriously ill patients. Consequently, there are strong justifications across the National Institutes of Health (NIH)'s entire purview for developing genetically modified and identical NHPs. Together, this applied research will develop protocols for routinely propagating cloned and transgenic NHPs of vital importance for clinically relevant research.
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