Lipofection with Lipofectamine™ 2000 in a heparin-free growth medium results in high transfection efficiency in chicken primordial germ cells

Tenkai Watanabe, Yuta Ochi, Ryota Kajihara, Kennosuke Ichikawa, Ryo Ezaki, Mei Matsuzaki, Hiroyuki Horiuchi

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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 was 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. This article is protected by copyright. All rights reserved.

Original languageEnglish
Article numbere2300328
Pages (from-to)1-40
Number of pages40
JournalBiotechnology Journal
Volume18
Issue number12
Early online date10 Aug 2023
DOIs
Publication statusPublished - Dec 2023

Keywords / Materials (for Non-textual outputs)

  • chicken primordial germ cells
  • lipofection
  • genome editing
  • knock-in strategy
  • CRISPR/Cas9

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