Pcmv sport6 vector

Full-length mouse Serpinb9 cDNA was amplified from the pCMV-SPORT6 vector (Dharmacon, accession BC029900, clone 4925100) using primers (forward, 5′-GCTCTAGAATGAATACTCTGTCTGAAGG-3′, reverse, 5′-CGGGATCCTGGAGATGAGAACCTGCCAC-3′) and inserted into the XbaI and BamHI sites of pCDH-CMV-MCS-EF1-Hygro vector (System Biosciences). The cloning of the vector was validated by sequencing. This vector together with pMD2.G and pCMV-dR8.74 were used for transfection of 293T cells to produce lentiviral particles. Viral particles collected from the culture supernatants were used for transfection of cancer cells using SureENTRY.

Q67L and T22N mutant Rab7A fragments were amplified from pET28 Rab7A constructs (obtained from Christopher Burd) and cloned into the pT_puro vector at the BamHI and EcoRI sites. The wild-type Rab7B fragment was amplified from Rab7B cDNA in pCMV-SPORT6 vector (Dharmacon) and cloned into the pT_hygro vector (Clontech #631034) at the BamHI and EcoRI sites. The Q67L and T22N mutant Rab7B fragments were generated by PCR-directed mutagenesis of pT_hygro-Rab7B construct. Retrovirus expressing each construct was produced in 293T cells as described above. HeLa S3 cells were transduced with Rab7A retroviruses and selected with puromycin or with Rab7B retroviruses and selected with hygromycin. To generate stable cell lines co-expressing Rab7A and Rab7B mutants, HeLa-tTA cells were sequentially transduced with Rab7A retrovirus and selected with puromycin and then transduced with Rab7B retrovirus and selected with hygromycin.