Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system

Abstract

Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.

Efficient TALEN-mediated gene knockout in livestock

“…The combination of TALENs plus the transposon components had no apparent impact on the utility of cells for cloning. We achieved a pregnancy rate of 78% with mono-and biallelic modified Ossabaw fibroblasts, a rate similar to our previous results with transposon transgenic Landrace cells (23). This result is especially encouraging considering that the cloning of Ossabaw swine, which are superior models of metabolic syndrome, had not previously been reported.…”

“…In contrast, transposon coselection allowed efficient isolation of stable cells containing mono-and biallelic indels without FACS or a physically linked selectionmarker. These targeted modifications can be easily segregated away from the selection transposons in a single generation of breeding during line propagation (23).…”

“…Although an average genomic modification level of 25% (i.e., ∼44% of cells with at least one modified allele; calculations described in Table 1) would be reasonable for direct somatic-cell nuclear transfer of cell populations, a method to enrich for modified cells or isolation of specific modified clones would be useful. To this end, we evaluated the stability of cells containing indels after an extended period in culture and tested an indel enrichment strategy based on transposon coselection (23). To apply this approach to TALEN-gene modification, a Sleeping Beauty transposon carrying a selection marker plus SB100X transposase was added to transfections with TALEN-encoding plasmids at a ratio of 1:5.…”

“…We have previously shown that transgenic primary fibroblast colonies can reliably be isolated and expanded when plated with wild-type fibroblasts and subjected to drug selection using transposon coselection (23). To evaluate this approach, we isolated puromycin-resistant colonies from cells treated with six TALEN-pairs and evaluated their genotypes by the Surveyor assay or direct sequencing of PCR products spanning the target site ( Table 1).…”

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…As an alternative to the direct embryo injection described here, both the SB [50][51][52] and the piggyBac 53 transposon systems have been used to genetically modify porcine cells in vitro, which were subsequently used as donors in SCNT for pig transgenesis.…”