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. 2016 Oct 21;26(11):1182–1196. doi: 10.1038/cr.2016.118

Figure 2.

Figure 2

Neural-specific knockout of mitochondrial genes induces cell non-autonomous UPRmt. (A) Targeting sequences for CRISPR-Cas9 knockout of spg-7 or cco-1, together with their PAM sequences. (B) Knockout of spg-7 in intestine activates hsp-6p::GFP reporter cell autonomously. odr-1p::dsRed is used as the co-injection marker. (C) Deletions of spg-7 or cco-1 by CRISPR/Cas9 are detected by T7E1 assay. Representative DNA gels of T7E1 assay show spg-7 or cco-1 PCR products amplified from genomic DNA of hsp-6 p::GFP worms (control), or unc-119p::Cas9+u6p::spg-7-sg(left), rab-3p::Cas9+u6p::cco-1-sg(right) worms. (D) Neural-specific knockout of spg-7 or cco-1 activates hsp-6p::GFP in distal tissues. (E) Western blotting shows the increasing level of hsp-6p::GFP in rab-3p::Cas9+u6p::spg-7-sg strain. The control lysate is from hsp-6p::GFP strain. Cell lysates are probed with GFP antibody. Tubulin is used as a loading control. (F, G) Neural knockout of spg-7 fails to induce UPRER or HSR in distal tissues. As positive controls, tunicamycin treatment robustly induces hsp-4p::GFP reporter, heat shock 1 h at 34 °C robustly induces hsp-16.2p::GFP reporter.