Pbmn ires gfp

pBMN-IRES-GFP was purchased from Addgene and the GFP was replaced by a truncated humanCD2 fragment to generate pBMN-IRES-hCD2, a Flag sequence was then inserted in front of the multiple cloning site of pBMN-IRES-hCD2 to generate pBMNFlag-IRES-hCD2. Full-length mouse PAXX was inserted into the EcoRI digested pBMNFlag-IRES-hCD2. pBMN-XLF-IRES-hCD2 and pBMN-FLAG-KU80-IRES-hCD2 and pBMN-FLAG-KU80ΔCTD-IRES-hCD2 were described previously3 (link)13 (link).

The hPTF1A promoter-HA-K-ras^G12D-IRES-EGFP-hygro expression vectors were modified from pCAGGS-hygro, pBMN-IRES-GFP (#1736, Addgene, Cambridge, MA, USA), and pBabe-K-ras^G12D (#58902, Addgene) respectively, by replacing the CMV early enhancer/chicken β actin (CAG) promoter between Sal I and Nhe I restriction sites with human PTF1A 3kb promoter, which was PCR amplified from human genomic DNA.
To immortalized primary miniature pig PCs (iPCs), PC cells were transfected with pBabe-puro SV40 LT (#13970, Addgene) retrovirus. After 48 h, cells were treated with 5 μg/mL puromycin for selection. iPCs were transfected with the hPTF1A promoter-HA-K-ras^G12D-IRES-EGFP-hygro vector and selected using 500 µg/mL hygromycin to create KiPC cell lines stably expressing K-ras^G12D. Subsequently, the cell sorting process is applied to the GFP-labeled single cell by flow cytometer (BD FACSAria™ III, BD Biosciences, San Jose, CA, USA) to obtain 100% clonal purity. Each line was evaluated by Western blotting and immunocytochemistry to confirm efficient overexpression of K-ras^G12D.