GGTA-1 targeting efficiency with a xenograft transgene

Abstract

Gene targeting by homologous recombination was utilized to target the GGTA-1 locus. Two independent enrichment strategies were evaluated to determine if targeting efficiencies at the GGTA-1 locus could be improved. The first strategy compared introduction of exogenous plasmid DNA in either single-stranded or double-stranded conformations. The second strategy utilized a promoter-trap vector and evaluated if further enrichment could be achieved with the addition of a negative selectable marker, a truncated diphtheria toxin cassette (tDT). These studies demonstrated for the targeting vector used herein, single-stranded DNA and double-stranded DNA yielded similar targeting efficiencies. In addition, tDT inclusion within the vector or as a co-transfectant did not enrich gene targeting provided by the promoter trap strategy alone. The strategies evaluated within were able to achieve a targeting frequency of 13% with a targeting efficiency of 1.5 X $105$ to $2.5 X 106$. Additionally, this is the first study to report the insertion and expression of a human transgene, hCD55, in the porcine GGTA-1 locus. Subsequent offspring had prominent expression of hCD55 in all tissues examined. The data combined suggests that the GGTA-1 locus can be targeted at a high frequency and the GGTA-1 locus could be a safe-harbor for transgenes.