Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs

Abstract

Genetically engineered pigs are often created with a targeting vector that contains a loxP flanked selectable marker like neomycin. The Cre–loxP recombinase system can be used to remove the selectable marker gene from the resulting offspring or cell line. Here is described a new method to remove a loxP flanked neomycin cassette by direct zygote injection of an mRNA encoding Cre recombinase. The optimal concentration of mRNA was determined to be 10 ng/μL when compared to 2 and 100 ng/μL (P < 0.0001). Development to the blastocyst stage was 14.1% after zygote injection with 10 ng/μL. This method successfully removed the neomycin cassette in 81.9% of injected in vitro derived embryos; which was significantly higher than the control (P < 0.0001). Embryo transfer resulted in the birth of one live piglet with a Cre deleted neomycin cassette. The new method described can be used to efficiently remove selectable markers in genetically engineered animals without the need for long term cell culture and subsequent somatic cell nuclear transfer.