FACS-based strategy to generate a CENP-A knockout allele.

<p>(A) Schematic to scale outlining targeting strategy. Correct targeting of the rAAV transduced construct results in the in-frame fusion of EYFP after the first codon of exon 3 and the deletion of the remainder of exon 3 and exon 4. polyA: SV40 poly adenylation signal. (B) Targeting of CENP-A-EYFP knockout (CAtail-EYFP) construct into human hTERT-RPE cells. FACS profiles of the 1^st (enrichment) and 2^nd (purification) runs are shown. Gates used to collect EYFP positives are indicated. Number of cells collected per total number of cells analyzed is indicated for each run. (C) Live cell differential interference contrast (DIC) and epifluorescent (EYFP) image is shown for a representative monoclonal isolate. Note the loss of centromeric signal and resulting diffuse nuclear EYFP fluorescence. (D) PCR verification of CENP-A knockout. Left and right flanks were analyzed analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032646#pone-0032646-g003" target="_blank">Figure 3B</a>. Right panel (across): A PCR using two primers flanking the targeted domain resulting in the amplification of both the targeted knockout allele (KO) as well as the untargeted wild type allele (WT). Sizes of marker fragments (M) and of expected products are indicated. All primer numbers correspond to those listed in methods (E) Immunoblot showing targeted CENP-A knockout clone and parental wild type hTERT-RPE cells. Anti-CENP-A antibody reveals a novel high molecular weight species in which a 70 amino acid N-terminal fragment of CENP-A is fused to EYFP (CAtail-EYFP).