Construction Of Technology Platform For Producing Gene-modified Rabbit

Rabbit is a traditional animal species used in biological studies. Compared to mouse, rabbit has a relatively larger body size and blood volume, making it suitable for studies of the cardiovascular, pulmonary, eye, and metabolism systems. In addition, rabbit is also widely used in pharmaceutical industry.Gene-modified new rabbit strains should find wide applications in biomedical research and bio-industry. Rabbits with modifications in specific genes will provide animal models for human diseases, and be valuable for studying diseases including atherosclerosis, cancer, AIDS and hypertrophic cardiomyopathy. Gene-modified rabbits will also meet specific requirement and be useful in toxicity tests and drug screen. Through gene modification, rabbits can be transformed into bioreactors to produce proteins of therapeutic and/or commercial values.Our laboratory has successfully established the technologies to derive rabbit embryonic stem cells and to clone rabbits using either somatic cells or embryonic stem cells as nuclear donors. When combined with gene-modifying approaches, the technology platform should allow us to produce various new rabbit strains with desired biological traits for a variety of applications. Work presented in this thesis records our effort towards this goal, focusing in technology development aiming to modify the rabbit genome.The thesis is divided into three parts. First, molecular cloning of the rabbit HPRT gene using the recombineering system. We constructed a knockout HPRT targeting vector by replacing the last three exons with a PGK-Neo-bGHpA cassette; we also constructed a promoterless knockin targeting vector by inserting a IRES-EGFP-IRES-Neo-bGHpA cassette in the 3'UTR of the HPRT gene. Second, molecular cloning of the rabbit POU5F1 gene and constructed a promoterless knockin targeting vectors by inserting a IRES-EGFP-IRES-Neo-bGHpA cassette in the 3'UTR of the POU5F1 gene. In the last part, rabbit fibroblast cells expressing red fluorescent protein (RFP) was used as nuclear donor cells to produce cloned embryos. Results from this experiment demonstrated that the goal to produce gene-modified rabbit is achievable.