Abstract
ABSTRACTThe near-haploid human cell line HAP1 recently became a popular subject for CRISPR/Cas9 editing, since only one allele requires modification. Through the gene-editing service at Horizon Discovery, there are at present more than 7500 edited cell lines available and the number continuously increases. The haploid nature of HAP1 is unstable as cultures become diploid with time. Here, we demonstrated some fundamental differences between haploid and diploid HAP1 cells, hence underlining the need for taking control over ploidy status in HAP1 cultures prior to phenotyping. Consequently, we optimized a procedure to determine the ploidy of HAP1 by flow cytometry in order to obtain diploid cultures and avoid ploidy status as an interfering variable in experiments. Furthermore, in order to facilitate this quality control, we validated a size-based cell sorting procedure to obtain the diploid culture more rapidly. Hence, we provide here two streamlined protocols for quality controlling the ploidy of HAP1 cells and document their validity and necessity.This article has an associated First Person interview with the co-first authors of the paper.
Highlights
Mammalian somatic cells are usually diploid, containing one genome copy from each parent, while haploidy is typically confined to the germline (Wutz, 2014)
The difference in DNA-quantity between haploid and diploid HAP1 cells, demonstrate that ploidy status can be determined with propidium iodide (PI)-based flow cytometry
Once the gene-specific knockouts are achieved, and phenotypic analyses of knockout cells are to be compared to parental control cells, it is of great importance to be aware of ploidy instability as a potential source of interfering variables
Summary
Mammalian somatic cells are usually diploid, containing one genome copy from each parent, while haploidy is typically confined to the germline (Wutz, 2014). Aberrant chromosome loss may occur during tumorigenesis (Zasadil et al, 2013) and rare human cancers with a hypodiploid karyotype have been found, for example in leukemia (Oshimura et al, 1977; Andersson et al, 1995). HAP1 is a near-haploid adherent fibroblast-like cell line of human leukemia origin. Such haploid cells are powerful tools for studying gene function as they contain a single copy of the genome and are unable to mask the effect of any mutations, which could possibly occur in +/− heterozygotes (Li and Shuai, 2017).
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