The Production Of Gene-Editing Rabbit Models Based On Cloning Technology

With the identification of more and more genomic sequences, great changes have been taken place in understanding of the biological nature, then how to use the information to study the function of these genes? In basic research, the common used methods of producing animal models are transgenic technology and gene targeting technology. In the study of producing genetically modified big animals, the production of transgenic and gene targeting animal models based on cloning technology plays an important role in the field of medicine and basic scientific research.The rabbit has long been used as a laboratory animal model for studying human disease, including cardiovascular, metabolic, and respiratory diseases, as well as various types of cancer. Rabbits offer several advantages over other laboratory animals: their larger size allows surgical procedures that could not be performed in mice or rats and they are phylogenetically closer to humans. In addition, in the field of human reproduction, high conservation of the genes indicates that the rabbit would be a valuable model for the study of embryonic preimplantation development; moreover, the embryo and feto-placental development of rabbit are similar to that in human. Nevertheless, there are only a limited number of transgenic or natural mutation rabbits for study, therefore the experimental animal model of rabbit with genome editing has extensive application prospects and theory value.Our work focused on the production of rabbit models with genetic editing, which is based on cloning technology, including two parts.(i) We constructed a reporter plasmid containing the gene encoding the enhanced green fluorescent protein(EGFP) under the control of the rabbit Oct4 promoter(prOG), and prOG transgenic rabbits were produced by somatic cell transfection and somatic cell nuclear transfer(SCNT) technology. Both prOG SCNT embryos and prOG F1 embryos that derived from a transgenic rabbit expressed EGFP. In addition, green fluorescence was readily detected in gonads isolated from 27 dpc fetuses. Then we cultured prOG-SSCs and prOG-ES cell lines, the green fluorescence were observed. And prOG-ES cells comprise a heterogeneous cell population, two different cell subpopulations were conducted with preliminary epigenetic analysis. The prOG transgenic rabbit represents a new model for studying the derivation and maintenance of rabbit pluripotent cells, which provides a useful tool to monitor pluripotency in rabbit pluripotent cells and may facilitate further studies on the regulation of rabbit embryogenesis.(ii) In this study, we described the production of HPRT gene knock-out rabbits by recombinant Adeno-associated virus(rAAV)-mediated homologous recombination and SCNT. For enhancing gene targeting rates, gene trap strategies were employed. Male and female gene knock-out fibroblast cell lines were derived by different strategies. When male HPRT knock-out cells were used for SCNT, no live rabbits were obtained. However, when female HPRT+/- cells were used for SCNT, live and healthy rabbits were generated. HPRT+/- cloned rabbits were fertile at maturity and already had stable genetic modified offspring. The methods we used provide a new idea for the low efficiency of cell lines in genetic manipulation and can be readily adapted to many other species and different genes.The two rabbit models that we constructed through cloning and genetic modification may provide an operational tool for genetic and developmental studies and human diseases.