Pcag hcas9 vector

The gRNA cloning vector and the pCAG-hCas9 vector were obtained from Addgene. The nucleotide sequence 5’-GAAGTTCTACATCCCTGGAGG-3’ in exon 2 of the human NDP52 gene was selected as the target. These plasmids and a puromycin-resistant vector (pXS-Puro) were co-transfected into HeLa cells, and puromycin-resistant cell clones were selected by limiting dilution. Genome editing of the NDP52 gene was screened by a BstNI digestion assay, and the mutations were confirmed by sequencing. The deficiency of the NDP52 protein was confirmed by immunoblotting.
For the knockout of the RNF31 gene, which encodes HOIP, the nucleotide sequence 5’-TCAACCCTCAGGAAGCTCAGC-3’ in exon 2 of the human RNF31 gene was selected as the target. Genome editing of the RNF31 gene was screened by a BtsCI digestion assay, and the mutations were confirmed by sequencing. The deficiency of the HOIP protein was confirmed by immunoblotting.

Stage 8-10 chick embryos were electroporated in the olfactory placode region, and stage 10/11 chick embryos were electroporated in the head ectoderm in and around the olfactory placodal region, or the prospective retina by applying three pulses (9-12 V, 25 ms duration, 1 s interval), adapted to previous experiences (Wittmann et al., 2014a (link)). Vectors used were: pCAβ-EGFPm5 (Yaneza et al., 2002 (link)), pCAG-hCas9 vector (addgene # 51142), pUC19-Sox2gRNA, pUC19-Cont-gRNA, pCAGGS-Hes5 (Holmberg et al., 2008 (link)) and pMiwIII–Noggin (Timmer et al., 2002 (link)), all at a concentration of 1.0 μg/μl (Timmer et al., 2002 (link)). Inhibition of BMP signalling by the Noggin construct has previously been verified (Maier et al., 2010 (link); Pandit et al., 2011 (link)). The constructs were transferred using an Electro Square Porator ECM 830 (BTX). After electroporation, the eggs were re-incubated to approximately stage 20-22 (olfactory epithelium) or stage 24 (retina). Viable embryos with GFP expression in the region of interest were selected for further analysis.