327 HIGH EFFICIENCY SWINE CLONING USING MONOGENIC AND POLYGENIC POOLS OF GENETICALLY MODIFIED CELLS

Abstract

Somatic cell nuclear transfer (SCNT) of genetically modified (GM) cells is currently the most widely applied method for the creation of transgenic swine. However, significant clone-to-clone variation in the efficiency of SCNT for a variety of genetically modified cell lines is commonly observed and contributes to the expense of transgenic animal production. A retrospective look at our own results based on the use of monoclonal GM cell lines as donors revealed a dismal efficiency of only 13% (15 embryo transfers resulting in only 2 full term pregnancies). Thus, while SCNT of individual GM clones offers the perceived advantage of prior characterisation of transgene expression or structure, the variability of clonability for any given cell line adds risk to SCNT. In contrast, rates of pregnancy when we used pools of GM cells as donors for SCNT were much better (9 full term pregnancies from 11 transfers, ∼82% efficiency). Four of these litters were derived from polyclonal but monogenic GM cell populations constitutively expressing either human APOBEC3G or YFP-Cre transgenes integrated using the Sleeping Beauty transposon system. Four litters relied on a novel approach, wherein different polyclonal and monogenic GM cell populations (containing different transgenes) were mixed before being used as donors for SCNT. For example, 2 litters were derived from the pooling of 2 GM cell populations carrying different tetracycline inducible or repressible shRNA transgenes, resulting in founders harboring each of the shRNA transgenes (4 in total). Two litters were also created from a pool of 3 distinct polyclonal cell populations, each harboring a different Cre-lox regulated transgene, resulting in the birth of 11 live piglets with founders corresponding to each of the transgenes. Thus, both mono- and polygenic pooling of GM cells significantly enhances the success of SCNT, largely avoiding variation in cell clonability. Furthermore, pooling results in a significant reduction in the time and number of surrogates required to generate a diversity of genetically modified pigs. Importantly, the use of Sleeping Beauty transgene integration resulted in a high rate of transgene-expressing founders. Where expected, the gene of interest transgenes were expressed in 23 of 29 founders (79%), whereas selectable marker transgene expression was observed in 35 of 40 founders (88%). The combination of efficient SCNT from polyclonal and polygenic cell populations and the high proportion of expressers delivered by Sleeping Beauty transgene integration offer a high-efficiency, low-risk solution to swine transgenesis.