RRC ID 80079
Author Watanabe T, Ochi Y, Kajihara R, Ichikawa K, Ezaki R, Matsuzaki M, Horiuchi H.
Title Lipofection with Lipofectamine™ 2000 in a heparin-free growth medium results in high transfection efficiency in chicken primordial germ cells.
Journal Biotechnol J
Abstract Primordial germ cells (PGCs) that can differentiate into gametes are used to produce genome-edited chickens. However, the transfection efficiency into PGCs is low in chickens; therefore, the yield efficiency of PGCs modified via genome editing is problematic. In this study, we improved transfection efficiency and achieved highly efficient genome editing in chicken PGCs. For transfection, we used lipofection, which is convenient for gene transfer. Chicken PGC cultures require adding heparin to support growth; however, heparin significantly reduces lipofection efficiency (p < 0.01). Heparin-induced lipofection efficiency was restored by adding protamine. Based on these results, we optimized gene transfer into chicken PGCs. Lipofectamine 2000 and our PGC medium were the most efficient transfection reagent and medium, respectively. Finally, based on established conditions, we compared the gene knock-out efficiencies of ovomucoid, a major egg allergen, and gene knock-in efficiencies at the ACTB locus. These results indicate that optimized lipofection is useful for CRISPR/Cas9-mediated knock-out and knock-in. Our findings may contribute to the generation of genome-edited chickens and stimulate research in various applications involving them.
Volume 18(12)
Pages e2300328
Published 2023-12-1
DOI 10.1002/biot.202300328
PMID 37559489
MeSH Animals CRISPR-Cas Systems* / genetics Chickens* / genetics Gene Editing / methods Germ Cells Heparin Transfection
IF 3.912
Resource
Chicken / Quail