Journal: Scientific Reports

Article Title: A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

doi: 10.1038/srep44816

Figure Lengend Snippet: ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the plentiCRISPR loxP vector plasmid DNA. BsmBI cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.

Article Snippet: To create the A 2A R knockout line (A2AR KO), a viral vector was created using the plentiCRISPR loxP plasmid , which was modified from the original plasmid plentiCRISPR V2 (Addgene plasmid # 173707) by addition of a loxP site in the 3′LTR .

Techniques: Sequencing, Plasmid Preparation, Construct, CRISPR, Marker

Journal: Scientific Reports

Article Title: A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

doi: 10.1038/srep44816

Figure Lengend Snippet: ( a ) Flow cytometry for Cas9 expression in K562 cells before and after transduction with the plentiCRISPR loxP A2AR. After puromycin selection to remove non-transduced cells, 96% of cells were successfully transduced. ( b ) Flow cytometry for removal of the loxP cassette using the pcDNA3.1 ZEO T2A CRE-GFP plasmid. 32% of cells express GFP, indicating successful removal of the proviral vector cassette. These cells were further selected using zeocin for isolation of cassette free cells. ( c ) Left, RT-PCR of clones selected from loxP cassette-free A 2A R knockout K562 cells. Right, sequencing of clone 12 reveals a one base (T) insertion on one allele and an 18 base deletion on the other for homologous knockout of the A 2A R gene.

Article Snippet: To create the A 2A R knockout line (A2AR KO), a viral vector was created using the plentiCRISPR loxP plasmid , which was modified from the original plasmid plentiCRISPR V2 (Addgene plasmid # 173707) by addition of a loxP site in the 3′LTR .

Techniques: Flow Cytometry, Expressing, Transduction, Selection, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Knock-Out, Sequencing