Journal: Matrix biology : journal of the International Society for Matrix Biology
Article Title: CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention
Figure Lengend Snippet: Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
Article Snippet: To identify the mutation in the RCS-Cas9 clone #7, the same PCR product was also amplified but with a different DNA polymerase, TaKaRa LA Taq® DNA polymerase (Clontech), and this PCR product cloned into the pCR4-TOPO vector (Invitrogen) according to the manufacturer's instructions.
Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Purification, Agarose Gel Electrophoresis, Labeling, Clone Assay, Genomic Sequencing