Pcmv cat t7 sb100x

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

All the synthetic oligonucleotides used as primers were purchased (Integrated DNA Technologies, Tokyo, Japan). The coding-fragment of the RBM8A protein was amplified by PCR on cDNA prepared from MCF-7 cells using KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan) with the specific primer set (Table S3). The amplified DNA was subcloned into a TA cloning vector pGEM-T (Promega, Madison, WI, USA) using TaKaRa Ex Taq polymerase (Takara Bio, Kusatsu, Japan). The obtained pGEM-T-RBM8A plasmid was cleaved with Sfi I and subcloned into the Sfi I site of the pSBtet-GP vector (Addgene, Watertown, MA, USA) [25 (link)].
In order to establish stable cell lines, the pSBtet-GP constructs (pSB-DsRed and pSB-RBM8A) were co-transfected with the Sleeping Beauty transposon vector pCMV(CAT)T7-SB100X (Addgene) [26 (link)] using Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. At 48 h post-transfection, cells were selected with 1 µg/mL puromycin (Sigma-Aldrich) for ~10 days and terminated when most cells showed GFP-derived green fluorescence. To induce RBM8A and the control DsRed proteins, 50 ng/mL of doxycycline (Takara Bio USA, Mountain View, CA, USA) was added to each cell line and cultured for 48 h.

DNA preparation for SB containing yellow fluorescence protein (YFP) and SB100X transposase were reported previously. The transposase plasmids for SB (pCMV(CAT)T7-SB100X) and PB (pCy43) were purchased from Addgene (http://www.addgene.org, Plasmid#34879) and provided by Sanger Institute (Hinxton, UK). Rox-GFP-polyA-rox and RFP were amplified by gateway PCR cloning (MultiSite Gateway^® Pro Plus, Invitrogen, 12537100, Life Technologies, Carlsbad, CA, USA) and inserted into final expression vector, PB-CAG (http://www.addgene.org/, #20960). Beta-Casein promoter and hIL2 cDNAs were amplified by PCR and inserted into PB-GFP by Infusion Cloning (In fusion HD cloning kit, Clontech, 639644, California, US). All the DNA vectors used in this study were illustrated in Supplementary Figure 1.