RRC ID 55990
Author Ishino T, Hashimoto M, Amagasa M, Saito N, Dochi O, Kirisawa R, Kitamura H.
Title Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation.
Journal Biomed Res
Abstract Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.
Volume 39(2)
Pages 95-104
Published 2018
DOI 10.2220/biomedres.39.95
PMID 29669988
MeSH Animals CRISPR-Cas Systems Cattle Clone Cells Fibroblasts / metabolism* Gene Editing* / methods Genetic Loci Genotype HeLa Cells Humans Mutation Nuclear Transfer Techniques* Receptor-Like Protein Tyrosine Phosphatases, Class 2 / genetics Reproducibility of Results Transfection
IF 1.217
Times Cited 5
Resource
Human and Animal Cells HeLa