Figure 2. 6F2 cells are haploinsufficient for rpL13a.

(A) Genomic DNA isolated from WT and 6F2 mutant cells was digested with Bgl II, Nco I, and Xba I and analyzed by Southern blotting with ³²P-labeled probe derived from ROSAβgeo retroviral sequences. (B) Upon transduction with the ROSAβgeo retrovirus, the provirus containing the splice acceptor (SA), the promoterless β-galactosidase-neomycin resistance cassette (βgal neo^R), and polyadenylation (polyA) sequences integrated into the rpL13a gene in 6F2 cells. Diagram shows resulting ROSAβgeo fusion transcript containing 5′ end of rpL13a mRNA and βgal neo^R sequences and the endogenous WT rpL13a mRNA. Forward (L13a 5′) and reverse (L13a 3′) primers for rpL13 and reverse proviral primer (βgeo 3′) are shown with arrows. (C) PCR was performed with the indicated primers on cDNA from WT and 6F2 cells to detect endogenous rpL13a and fusion transcript expression. (D) Basal rpL13a RNA expression in WT and 6F2 mutant cells was determined by qRT-PCR and normalized to β-actin expression. (E) Basal rpL13a protein expression in WT and 6F2 mutant cells was determined by western blotting and quantified by densitometry. Representative blot shown for rpL13a and actin loading control. All data expressed as mean ± SE for 3 independent experiments. * p < 0.01 for mutant 6F2 vs. WT. (See also supplemental Figure 2.)