Figure 5

Cre-regulated perturbation of Bap1, Pbrm1, and Setd2 in BPS-Cre mice. (A) For the initial targeting of mouse Bap1, Pbrm1, and Setd2 we employed a dox-responsive GFP-IRES-Cas9^D10A allele, whereas in a revised approach we employed a cre-regulated Cas9-P2A-GFP (left and right, respectively). Ggt promoters drive tissue specific expression of the Cas9 activator (tTA and Cre, respectively), and facilitated gene perturbation specifically in the proximal tubules of the kidneys of the TRACK ccRCC mouse model (BPS-TA and BPS-Cre animals). (B) Immunostaining of GFP served as a marker for proximal tubule cells with active Cas9. Note the more pervasive expression using tTA regulation compared with Cre regulation (left and right, respectively). Magnification; × 200, scale bar; 100 μm. Representative images are shown. (C) Co-immunostaining of GFP and the Flag epitope of Cas9 (green and red, respectively) demonstrates co-expression of the two proteins, and validates GFP as a proxy for Cas9 expression. Magnification; × 200, scale bar; 100 μm. Representative images are shown. D) Frequencies of on-site deletions in Bap1, Setd2, and Pbrm1 in BPS-TA and BPS-Cre kidneys. WT kidneys served as negative controls. Note that average deletion frequencies were increased in BPS-Cre vs BPS-TA kidneys for Pbrm1 (12.2% vs. 4.3%) and Setd2 (10.6% vs. 1.3%), whereas deletion frequencies for Bap1 was unchanged in BPS-Cre vs BPS-TA kidneys (0.9% vs. 0.9%), but higher than background levels (WT kidneys; 0.3%). Note also that one animal (#3) among the BPS-Cre animals had the highest levels of gene editing of Bap1, Pbrm1, and Setd2 (1.6%, 23.4%, and 20.7%, respectively). Note that all BPS-TA mice evaluated were biallelic for each of the three transgenes, and all BPS-Cre mice were biallelic for each of the four transgenes. The transgene overview and figure panels was generated and compiled in Microsoft Powerpoint software.