Method for producing interspecies transgenic animal using porcine oocyte

摘要

A method for producing an inter-species transgenic animal is provided to produce a transgenic pig through an inter-species somatic cell nuclear transfer technique using porcine oocytes. A method for producing an inter-species transgenic animal comprises the steps of: (a) integrating an enhanced green fluorescent protein(EGFP) gene into a retrovirus vector to construct an EGFP-retrovirus vector; (b) transfecting the EGFP-retrovirus vector into a target xenogenic primary fetal fibroblast; (c) recovering porcine oocytes and in vitro ripening the porcine oocytes; (d) denucleating the ripened porcine oocytes; (e) after integrating the fetal fibroblast including the EGFP-retrovirus vector into the denucleated oocytes, fusing and activating the oocytes; (f) in vitro culturing the activated porcine cytoplasm reconstructed by the xenogenic donor cell; and (g) investigating the EGFP gene expression of the reconstructed embryos using a fluorescence microscope or subjecting the reconstructed embryos to PCR analysis using a primer set specific to the EGFP gene, which is a primer pair described as SEQ ID : NOs. 5 and 6, and a primer set specific to the Y chromosome, which is a primer pair described as SEQ ID : NOs. 1 and 2, to identify the EGFP integration and clonality of the embryos, wherein the xenogenic is cattle, sheep, mouse or non-human primate excluding human. Further, the fusion is performed by inducing the oocytes for 30 seconds through 2.1 kV/cm of single direct current pulse.