Single-cell cloning of human T-cell lines reveals clonal variation in cell death responses to chemotherapeutics.
Cell death
Chemotherapeutic resistance
Cloning
Leukemia
T-ALL
Journal
Cancer genetics
ISSN: 2210-7762
Titre abrégé: Cancer Genet
Pays: United States
ID NLM: 101539150
Informations de publication
Date de publication:
09 2019
09 2019
Historique:
received:
09
01
2019
revised:
18
04
2019
accepted:
09
06
2019
entrez:
27
8
2019
pubmed:
27
8
2019
medline:
3
4
2020
Statut:
ppublish
Résumé
Genetic modification of human leukemic cell lines using CRISPR-Cas9 has become a staple of gene-function studies. Single-cell cloning of modified cells is frequently used to facilitate studies of gene function. Inherent in this approach is an assumption that the genetic drift, amplified in some cell lines by mutations in DNA replication and repair machinery, as well as non-genetic factors will not introduce significant levels of experimental cellular heterogeneity in clones derived from parental populations. In this study, we characterize the variation in cell death of fifty clonal cell lines generated from human Jurkat and MOLT-4 T-cells edited by CRISPR-Cas9. We demonstrate a wide distribution of sensitivity to chemotherapeutics between non-edited clonal human leukemia T-cell lines, and also following CRISPR-Cas9 editing at the NLRP1 locus, or following transfection with non-targeting sgRNA controls. The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype. Whole genome sequencing of two clonal cell lines edited by CRISPR-Cas9 revealed unique and shared genetic variants, which had minimal read support in the non-clonal parental population and were not suspected CRISPR-Cas9 off-target effects. These variants included genes related to cell death and drug metabolism. The variation in cell death phenotype of clonal populations of human T-cell lines may be a consequence of T-cell line genetic instability, and to a lesser extent clonal heterogeneity in the parental population or CRISPR-Cas9 off-target effects not predicted by current models. This work highlights the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.
Identifiants
pubmed: 31447068
pii: S2210-7762(18)30569-6
doi: 10.1016/j.cancergen.2019.06.003
pmc: PMC6711394
mid: NIHMS1531657
pii:
doi:
Substances chimiques
Antineoplastic Agents
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
69-77Subventions
Organisme : NHLBI NIH HHS
ID : R01 HL124209
Pays : United States
Organisme : NCI NIH HHS
ID : R35 CA210030
Pays : United States
Organisme : NIAID NIH HHS
ID : R56 AI103352
Pays : United States
Organisme : Howard Hughes Medical Institute
Pays : United States
Informations de copyright
Copyright © 2019 Elsevier Inc. All rights reserved.
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