Extended Data Fig. 5: The generation of CRISPRa embryonic stem cell lines, and the evaluation of CRISPR-deletion mice.
From: Endogenous retroviruses drive species-specific germline transcriptomes in mammals
a, qRT-PCR analyses of CRISPRa embryonic stem (ES) cells show expression level changes of the dCas9-VPR transgene 24 h after doxycycline (Dox) induction. Expression levels were normalized to the endogenous housekeeping gene Hprt. Upon addition of Dox, all ES cell clones evinced overt dCas9-VPR mRNA expression. Because clone #6 exhibited the highest upregulation of dCas9-VPR transcript, we restricted further experiments to clone #6. b, Representative image of CRISPRa ES cell colonies at day 4 after transduction with the sgRNA lentiviral construct. We validated the degree of sgRNA expression through observations of the red fluorescent reporter protein DsRed. Scale bar, 200 µm. c, Testis sections from wild-type (WT; left) and Zfy2 enhancer-deletion mice (right) at postnatal day 28 (P28). The sections were stained with hematoxylin and eosin. Scale bars, 100 μm. In our observations of Zfy2 enhancer-deletion samples, we noted no gross changes to testis morphology; however, we observed multinucleated cells (arrowheads).
