FIG. 1.
Generation of a PRMT1 conditional allele in mice and PRMT1-deficient MEFs. (A) Schematic representation of the wild-type (PRMT1 locus), floxed (2 loxP; PRMT1FL), and null (1 loxP; PRMT1−) PRMT1 alleles. The exons are the black boxes, and the line represents introns not drawn to scale. The black triangles denote loxP sites, and the small arrows denote the primers (named CT#) used for PCR analysis. The expected size of the CT-497/498 DNA fragment for the wild-type allele is 245 bp, while the size of the DNA fragment for the 2loxP allele is 358 bp. The expected size of the CT-296/498 DNA fragment after CRE excision is 294 bp. (B) Primary MEFs deficient for PRMT1 were generated by infecting cells with a hygro-CRE retrovirus. Genomic DNA from hygromycin-selected cells was analyzed by PCR, and the DNA fragments were visualized on an ethidium bromide-stained agarose gel. M denotes molecular mass markers of the 1-kb ladder (Invitrogen, Inc.). (C) PRMT1 MEFs of the indicated genotype infected with hygro-Cre or not were lysed, and the total cellular proteins were analyzed by immunoblotting with anti-PRMT1 and anti-α-tubulin antibodies, as a loading control. The migration of PRMT1 and α-tubulin is shown on the right with arrows, and the migration of the molecular mass markers is shown on the left in kilodaltons. (D) PRMT1FL/− MEFs were immortalized and stably transfected with a plasmid encoding the estrogen receptor-CRE fusion protein (ER-CRE). The cells, termed PRMT1FL/−;CreERT MEFs or control PRMT1+/+;CreERT MEFs, were then treated with OHT for 0, 2, 4, and 6 days, and genomic DNA was isolated and analyzed as in panel B. (E) Total cellular proteins from mock-treated (−) or OHT-treated (+) PRMT1FL/−;CreERT and PRMT1+/+;CreERT MEFs were analyzed by immunoblotting as described for panel C.