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.

Gene targeting in mouse embryos mediated by RecA and modified single-stranded oligonucleotides

“…The gene targeting by RecA and modified single-stranded oligonucleotides is convenient and precise. Furthermore, RecA was shown to protect ssDNA from degradation by providing a protective coating on DNA (Chow et al 1986) and to increase embryo survival and transgene integration frequencies after pronuclear microinjection of RecA-ssDNA complex (Maga 2001;Maga et al 2003), although Mason et al (2006) recently argued that a protein coating of singleand double-stranded DNA constructs produced no significant differences in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born.…”

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals

“…Initial experiments addressed the effect of a bacterial recombinase (RecA) [60] and Tn5 transposase [61] on mouse and livestock transgenesis [62,63]. Both enzymes were able to increase the proportion of live transgenic animals compared to classic pronuclear microinjection and ICSI-Tr methods [64], but seemed to suffer from sub-optimal activities of the employed enzymes [29,30].…”

Use of single stranded targeting DNA or negative selection does not provide additional enrichment from a GGTA1 promoter trap

“…To achieve enrichment based on a vector design, several strategies have been used: promotertrap (mouse, [18]; pig, [8]), poly(A) trap (mouse, [19]; pig, [13]), and positive-negative selection (mouse, [20]; pig, [21]). It has also been observed that single-stranded DNA (ss-DNA) can serve as substrate for HR [22,23], but may not randomly integrate, as efficiently as double-stranded DNA (ds-DNA) [24]. It is, therefore, possible that ss-DNA may participate in homologous recombination at the similar rates to ds-DNA, while producing fewer random integrations events.…”

“…The hypothesis was that DNA conformation would alter the ratio of targeted-integration versus random-integration. This hypothesis was based on the expectation that ss-DNA may produce fewer random integration events [24], and thus, provide enrichment, or may produce more targeting events [14], and thus, increase targeting efficiency.…”