Article Title: Expression of MLL-AF4 or AF4-MLL fusions does not impact the efficiency of DNA damage repair
Figure Lengend Snippet: CRISPR/Cas9-mediated generation of syngeneic HEK293 cells expressing a unique copy of MA4 or A4M in the AAVS1 safe harbor A. , F. Schematic representation of the donor vector used for insertion of the dTo-MA4 A. or A4M-GFP F. cassette into the AAVS1 locus. dTo, dTomato fluorescent protein; SD, splice donor; SA, splice acceptor; CAG, CMV early enhancer/chicken β actin promoter. Black (5′; junction) and green (3′; junction) arrows depict genomic location of primers used to confirm targeted integration. B. , G. Representative images of dTo-MA4- and A4M-GFP-expressing HEK293 cells after puromycin or G418 selection, respectively. C. , H. Targeted integration analysis of MA4 and A4M into the AAVS1 locus by PCR using primers specific for the 5ʹ (top panels) and 3ʹ (bottom panels) integration junctions. D. , I. RNA expression of MA4 D. and A4M I. in antibiotic-selected cells. E. , J. Homologous recombination confirmed by southern blot analysis after BglII digestion of genomic DNA from puromycin/G418-resistant clones using a MA4 probe E. or an AAVS1 exon2 probe outside the targeting construct J. . A 4Kb band represents a targeted integration of MA4 in PPP1R12C. The 8kb band corresponds to the targeted integration of A4M in PPP1R12C. Untargeted allele gives a 12Kb band J. . L. qPCR of the MA4 targets HOXA9 and PROM1 is comparable between HEK293 cells ectopically expressing MA4 upon CRISPR/Cas9-mediated genome edition (left panel) or lentiviral transduction (right panel) * p
Article Snippet: Guide RNA against AAVS1 intron 1 was constructed using the primers AAVS1 Fw 5ʹ-CACCGGGGCCACTAGGGACAGGAT-3ʹ and AAVS1 Rv 5ʹ-AAACATCCTGTCCCTAGTGGCCCC-3ʹ [ ]. pAAVS1.SA-2A-Puro-pA donor vector was obtained from Addgene (plasmid #22075). pAAVS1.SA-2A-Neo-pA was made by replacing puro gene from pAAVS1.SA-2A-Puro-pA using Xho I and Mfe I restriction sites.
Techniques: CRISPR, Expressing, Plasmid Preparation, Selection, Polymerase Chain Reaction, RNA Expression, Homologous Recombination, Southern Blot, Clone Assay, Construct, Real-time Polymerase Chain Reaction, Transduction