The effect of coating single- and double-stranded DNA with the recombinase A protein of Escherichia coli on transgene integration in mice

Embryo survival and transgene integration rates are two major factors that influence the efficiency of transgenic animal production by pronuclear microinjection. Recombinase A protein-coated transgenes were compared for transgene integration and embryo survival with their non-coated counterparts in both single- and double-stranded forms. Murine zygotes were microinjected with a large 30 kb α_S1-casein/human lysozyme DNA construct and a small 5.5 kb β-lactoglobulin/desaturase DNA construct using four different construct preparations for each gene. The preparations included recombinase A protein-coated, single- and double-stranded DNA constructs and non-coated, single- and double-stranded DNA constructs. Using conventional non-coated, double-stranded DNA constructs, we obtained a transgene integration efficiency of 1.5% (1352 embryos transferred produced 20 transgenic pups). The same double-stranded DNA constructs coated with recombinase A protein yielded a similar percentage of transgene integration (1.1%, 18/1697). Using single-stranded DNA, non-coated constructs produced a transgene integration rate of 0.5%, while none of the 1040 zygotes injected with recombinase A-coated constructs produced transgenic pups. While recombinase A protein coating produced no effect on embryo survival, litter size or pregnancy rate with double-stranded constructs, a detrimental effect was observed on embryo survival (P < 0.001) and pregnancy rate (P < 0.005) with recombinase A protein coating of single-stranded human lysozyme DNA constructs. A trend toward increased embryo survival (P = 0.054) with no difference in pregnancy rate (P > 0.05) was observed with the recombinase A protein coating of single-stranded desaturase constructs. These results suggest that recombinase A protein coating of single- and double-stranded DNA constructs produced no significant differences (P > 0.05) in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born.