title: The effect of coating single- and double-stranded DNA with the recombinase A protein of Escherichia coli on transgene integration in mice.
abstract: 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 alpha(S1)-casein/human lysozyme DNA construct and a small 5.5 kb beta-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.
authors.affiliation: Department of Animal Science, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Mason
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Najarian
authors.forename: Gary B
authors.initials: GB
authors.lastname: Anderson
authors.forename: James D
authors.initials: JD
authors.lastname: Murray
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Maga
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics*; D004277:DNA, Single-Stranded; D004622:Embryo, Mammalian; D029968:Escherichia coli Proteins; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D009113:Muramidase / Q000235:genetics; D045522:Recombinases / Q000494:pharmacology*; D019076:Transgenes / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D045522:Recombinases; D004247:DNA; D009113:Muramidase