Article Title: Functional Interrogation of HOXA9 Regulome in MLLr Leukemia via Reporter-based CRISPR/Cas9 screen
Figure Lengend Snippet: Establishment and characterization of the HOXA9 P2A-mCherry reporter human MLLr leukemia cell line (A) Schematic diagram of the knock-in design and genotyping PCR primer design for the HOXA9 P2A-mCherry reporter allele. (B) Flow cytometry analysis of HOXA9 P2A-mCherry reporter cells. Wild-type SEM cells were used as negative controls. (C) Genotyping PCR products from the 5′ and 3′ knock-in boundaries were sequenced to verify the seamless knock-in of the mCherry reporter gene to the endogenous locus. (D) Fluorescence in situ hybridization of the P2A-mCherry knock-in cassette in HOXA9 P2A-mCherry reporter cells. The P2A-mCherry DNA was labeled with a red-dUTP by nick translation, and an HOXA9 BAC clone was labeled with a green-dUTP. The cells were then stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. A representative metaphase cell image is shown for the pattern of hybridization (pairing of red and green signals). (E) RNA-seq data of all HOXA cluster genes were illustrated as log 2 (normalized numbers of FPKM) from two replicate samples of SEM cells. HOXA7 , HOXA9 and HOXA10 were highlighted by color code. (F) Q-PCR analysis confirmed the unaffected HOXA cluster gene transcription between HOXA9 P2A-mCherry reporter (KI) and WT SEM cells. Data shown are means ± SEM from replicate independent experiments. *p
Article Snippet: Vector construction A pair of oligomers containing a 20-bp sgRNA (5’-AAAGACGAGTGATGCCATTT-3’) sequence targeting the surrounding genomic segment of HOXA9 stop codon was synthesized (Thermo Fisher Scientific) and cloned into the all-in-one vector, pSpCas9(BB)-2A-GFP (Addgene #48138) between BsmBI sites.
Techniques: Knock-In, Polymerase Chain Reaction, Flow Cytometry, Fluorescence, In Situ Hybridization, Labeling, Nick Translation, BAC Assay, Staining, Hybridization, RNA Sequencing Assay