Transposon Systems for Bovine Cell Engineering

In previous studies, the transposon system vectors for SB (pCMV (CAT)T7-SB100X) and PB (pCy43 and PB-CA) were purchased from Addgene (http://www.addgene.org, Plasmids #34879 and #20960, respectively), and/or provided by the Sanger Institute (Hinxton, UK) for the PB and SB systems [28 (link), 29 (link)]. To establish a novel Tol2 system, the transposon and transposase were purchased from Addgene (http://www.addgene.org, Plasmids #97151 and #31823, respectively). To avoid the potential backbone sequences effect in the application of the transposon systems, all transposon element sequences were amplified by PCR, and the PCR products were run for 15 min before being extracted with a Qiagen Gel Extraction Kit (Cat No. 28704). The extracted PCR products were cloned with a Qiagen TA cloning kit (Cat No. 231124) (Fig. 1A). All the reconstructed vectors were sequenced fully.Fig. 1

Schematic design of the transposon systems (PB, SB, and Tol2) application in bovine somatic and germ cells. A Illustration of the transposon DNA composition including the Ef1α promoter and GFP reporter gene. B Transfection and analysis method for the somatic cells, and C) germ cells