Application of transposon systems in the transgenesis of bovine somatic and germ cells

Abstract

Background Several DNA transposons including PiggyBac (PB), Sleeping Beauty (SB), and Tol2 have been applied as effective means for of transgenesis in many species. Cattle are not typically experimental animals, and relatively little verification has been presented on this species. Thus, the goal here was to determine the applicability of three transposon systems in somatic and embryo cells in cattle, while also investigating which of the three systems is appropriate for each cell type. Green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, the GFP-positive cells or blastocysts were observed through fluorescence, while the transfection efficiency was calculated by FACS. Results In bovine somatic cells, the PB (63.97 ± 11.56) showed the highest efficiency of the three systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Conversely, Tol2 (75.00%) and SB (70.00%) presented a higher tendency in the embryonic cells compared to PB (42.86%). Conclusions These results demonstrate that these three transposon systems can be used in bovine somatic cells and embryos as gene engineering experimental methods. Moreover, they demonstrate which type of transposon system to apply depending on the cell type.

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

“…Although a study reported that Tg rats can be produced more efficiently (33%–100%, mean of nearly 80%) using mPBase mRNA pronuclear microinjection 50 , it may be possible to consistently produce Tg animals even more efficiently if mPBase mRNA is introduced at an earlier timing after in vitro fertilization. Transgenic technology is also important for livestock, such as cattle, pig, and sheep, but there are very limited reports on application of the piggyBac transposon system to their zygotes 51,52 . Somatic cell nuclear transfer is one of the most common methods to create Tg livestock 53,54 .…”

“…The copyright holder for this this version posted December 10, 2023. ; https://doi.org/10.1101/2023.12.10.570953 doi: bioRxiv preprint timing after in vitro fertilization. Transgenic technology is also important for livestock, such as cattle, pig, and sheep, but there are very limited reports on application of the piggyBac transposon system to their zygotes 51,52 . Somatic cell nuclear transfer is one of the most common methods to create Tg livestock 53,54 .…”