<p>(A) Selection of colorectal cancer cell lines. (B) Diagram of the targeting strategy. The endogenous LSD1 locus is shown, along with the AAV targeting vector and the targeted allele before and after Cre-mediated recombination. PCR primers P1∼P10 were described in Materials and Methods. Three STOP codons TGA were added at the end of the left HA to ensure premature termination of the transcript. L-ITR and R-ITR, left and right inverted terminal repeats, respectively; HA, homology arm; TK, TK promoter; Neo, neomycin resistance gene; polyA, polyadenylation signal. (C) Identification of LSD1 null cell lines by genomic PCR. PCR detected the WT and targeted alleles from the indicated cells using genomic DNA as template. Two independent clones for the KO are shown. The lower row with the band designated “WT” indicates that the lower band was amplified from wild-type allele. The upper row with the band designated “KO” shows that the upper band was amplified from the allele with part of exon 7 of the LSD1 knocked out and LoxP inserted. (D) Confirmation of LSD1 null cell lines by Western blot analysis. β-actin was used as an internal control.</p
