Generation of a human induced pluripotent stem cell line via CRISPR-Cas9 mediated integration of a site-specific homozygous mutation in CHMP2B.
Authors:
Zhang Y, Schmid B, Nielsen TT, Nielsen JE, Clausen C, Hyttel P, Holst B, Freude KK.
In:
Source:
Stem Cell Res
Publication Date:
(
2016
)
Issue:
17(1)
:
151-153
Research Area:
Cancer Research/Cell Biology
Neurobiology
Stem Cells
Gene Expression
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
4D-Nucleofector® X-Unit
Experiment
iPSCs maintained on matrigel coated dishes (Corning Bioscience) in E8 medium(Gibco)were detached using Accutase (Gibco). 2 × 106 cells were co-transfected with 10 µg of the CRISPR-Cas9 plasmid (Addgene, 62,988) and 1 µL of the ssODNs.We used a 4D nucleofector (programme CA-167) from Amaxa in combination with the P3 Primary Cell Kit for transfection. iPSCs were subsequently transferred back to a matrigel coated dish in E8 medium supplemented with 1 mM ROCK inhibitor (Sigma). 24 h post-transfection, cells were subjected to puromycin selection for 48 h and allowed to recover for a week. Resistant colonies were picked and expanded for genotyping.
Abstract
Frontotemporal dementia (FTD) is an early onset neurodegenerative disease. Mutations in several genes cause familial FTD and one of them is charged multivesicular body protein 2B (CHMP2B) on chromosome 3 (FTD3), a component of the endosomal sorting complex required for transport III (ESCRT-III). We have generated an induced pluripotent stem cell (iPSC) line of a healthy individual and inserted the CHMP2B IVS5AS G-C gene mutation into both alleles, resulting in aberrant splicing. This human iPSC line provides an ideal model to study CHMP2B-dependent phenotypes of FTD3.
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