Figure 2.

HDR-mediated Venus knock-out using the SGD52 construct. The MEF cells carrying a single copy of Venus were electrotransfected with either pX459 plasmid expressing gRNA-252 and Cas9, or the SGD52 vector expressing gRNA-252 and Cas9 and puromycin plus an HDR cassette. The pSGD52-electrotransfected cells were selected against puromycin for 105 days. In parallel, cells from the pX459-252 and the non-transfected cells (positive control), expressing a single copy of Venus, were also cultured for 105 days. (A) Results of FACS analysis. The Venus expression pattern of PK and pX459/ pSGD52-electroporated cells are shown in red color. The MEF cells with no Venus transgene was considered as the negative control and was depicted in blue color. (B) Fluorescence microscopy and (C) PCR. Two sets of primers were used to amplify the Venus transgene and the Puromycin-Venus. Specific primers for Venus could detect only the wild type copy of Venus transgene under the CAGGS promoter in non-transfected cells, but not the truncated Venus using the SGD cassette. The presence of the HDR-mediated SGD cassette was verified using specific primers covering the part of the puromycin transgene and the proximal part of the truncated Venus. The SGD cells are electrotransfected cells with pSGD plasmids.