MSCV-based insertion mutagenesis in mESC

To perform insertional mutagenesis, we used MSCV to introduce Cre into the cells. This approach allowed us to perform insertional mutagenesis as well as delete the conditional allele in a single step. Stable virus-producing cells were generated by transfecting packaging cell line GP+E86 (ATCC, CRL 9642) with the plasmid. Transfected cells were selected with appropriate antibiotics (Hygromycin or G418). ES cells were transduced with the retrovirus in a 10 cm dish by coculturing PL2F7 mESC (1–2 × 10⁶) with packaging cells (3 × 10⁶) in the presence of 8 μg/ml polybrene (Santa Cruz Biotech) for 48 hr. Packaging cells were mitotically inactivated by mitomycin C (MMC) treatment (10 μg/ml for 2 hr). The transduced cells were washed with PBS, and then plated at lower density and selected for either in HAT medium or for antibiotic resistance. Individual colonies were picked into 96-well plates and further analyzed either by Southern hybridization as described previously (Kuznetsov et al. 2008). To identify the viral insertion site in rescued Brca2^ko/ko mESC, we used the Splinkerette PCR-based method (Li et al. 1999). We extracted genomic DNA from Brca2^ko/ko cells, digested with EcoRI, and ligated to the splinkerette oligos linker overnight. PCR was performed on the ligation reaction using gene-specific (Cre or Hygro) primers and splinkerette-specific primers followed by a nested PCR performed using primers recognizing the long terminal repeat of MSCV and the splinkerette linker. PCR products were loaded on 1% agarose gels. PCR bands were excised and purified using MiniElute columns (Qiagen, Valencia, CA), and sequenced directly using the Big Dye Cycle Sequencing kit (PerkinElmer, Shelton, CT) and an ABI Model 373A DNA Sequencer (Applied Biosystems, Foster City, CA).