HIGH EFFICIENCY METHODS AND COMPOSITIONS FOR INTEGRATING EXOGENOUS DNA INTO GENOMIC DNA OF SPERM

摘要

The present invention relates to high efficiency methods and compositions for stably integrating exogenous double-stranded DNA into the chromosomal DNA of sperm cells. The exogenous DNA to be integrated into the sperm is converted to a linear double stranded DNA possessing single-stranded cohesive ends, by contacting said exogenous DNA with a type II restriction enzyme that upon scission, generates such ends. The DNA to be cut can be a circular DNA such as in a plasmid, such that it possesses at least one recognition and cutting site outside of the genes or regulatory regions critical to the desired, post-integration function of the DNA, and no recognition and cutting sites within the critical regions. The exogenous DNA is then introduced into sperm cells, as is some of the type II restriction enzyme used above to generate the single-stranded cohesive ends on the exogenous DNA. The DNA and the corresponding restriction enzyme can be introduced into the sperm cells, together or sequentially, by electroporation or lipofection or by any other technique that preserves enough of said sperm's mobility and fertilization functions. The sperm containing the integrated exogenous DNA can then be used to fertilize eggs by artificial insemination, in vitro fertilization, or other fertilization methods known in the art, thereby producing transgenic animals wherein all or a plurality of cells now contain the exogenous DNA stably integrated into their respective genomes. The present invention also relates to transgenic animals or their descendants produced by methods or compositions herein disclosed. The present invention further relates to cells or cell lines derived from transgenic animals or their descendants produced by methods or compositions herein disclosed. The present invention further relates to proteins or other gene products encoded for by the exogenously introduced DNA isolated from said transgenic animals or their descendants. The present invention further relates to using the method of the present invention to produce transgenic animals resistant to diseases, and to the disease-resistant animals thereby produced.