CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

Autor: Mandl M; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria.; Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck , Austria., Ritthammer H; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria., Ejaz A; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria.; Adipose Stem Cell Center, Department of Plastic Surgery, University of Pittsburgh , PA, USA., Wagner SA; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria., Hatzmann FM; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria.; Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck , Austria., Baumgarten S; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria., Viertler HP; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria.; Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck , Austria., Zwierzina ME; Innsbruck Medical University , Innsbruck, Austria., Mattesich M; Innsbruck Medical University , Innsbruck, Austria., Schiller V; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria.; Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck , Austria., Rauchenwald T; Innsbruck Medical University , Innsbruck, Austria., Ploner C; Innsbruck Medical University , Innsbruck, Austria., Waldegger P; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria.; Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck , Austria., Pierer G; Innsbruck Medical University , Innsbruck, Austria., Zwerschke W; Division of Cell Metabolism and Differentiation Research, Research Institute for Biomedical Aging Research, University of Innsbruck , Austria.; Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck , Austria.
Jazyk: angličtina
Zdroj: Adipocyte [Adipocyte] 2020 Dec; Vol. 9 (1), pp. 626-635.
DOI: 10.1080/21623945.2020.1834230
Abstrakt: The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 ( SPRY1 ) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: In sertion/ Del etion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.
Databáze: MEDLINE
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