Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration

Ming Shi, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira

研究成果: ジャーナルへの寄稿学術誌査読

7 被引用数 (Scopus)

抄録

It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.

本文言語英語
ページ(範囲)363-370
ページ数8
ジャーナルJournal of Bioscience and Bioengineering
129
3
DOI
出版ステータス出版済み - 3月 2020

!!!All Science Journal Classification (ASJC) codes

  • バイオテクノロジー
  • バイオエンジニアリング
  • 応用微生物学とバイオテクノロジー

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