Generation of DAT-integrin α5 heterozygous knock-in embryonic stem cells using CRISPR/Cas9 system
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- Izumi Yasuhiko
- Lab. Pharmacol., Kobe Pharm. Univ.
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- Kinoshita Shinichi
- Dept. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ.
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- Fukuzawa Moeka
- Dept. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ.
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- Nishisako Kazuma
- Lab. Pharmacol., Kobe Pharm. Univ.
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- Ichimura Suzuka
- Lab. Pharmacol., Kobe Pharm. Univ.
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- Yamaki Kouya
- Lab. Pharmacol., Kobe Pharm. Univ.
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- Kume Toshiaki
- Dept. Appl. Pharm., Grad. Sch. Med. and Pharm. Sci., Univ. of Toyama
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- Koyama Yutaka
- Lab. Pharmacol., Kobe Pharm. Univ.
Bibliographic Information
- Other Title
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- CRISPR/Cas9システムを用いたDAT遺伝子へのインテグリンα5遺伝子ヘテロノックインマウスES細胞の作製
Abstract
<p>We have previously found that integrin α5β1 on dopaminergic neurons plays an important role in the neurite outgrowth on striatal neurons. This finding indicates that integrin α5 (Itga)-overexpressing dopaminergic neurons enhance functional regeneration in transplantation therapy for Parkinson disease. Here, we generated the dopamine transporter (DAT)-Itga heterozygous knock-in mouse embryonic stem cells using the CRISPR)/Cas9 system. These cells can be induced to express Itga gene after dopaminergic differentiation, avoiding the loss of DAT function. The knock-in targeting vector expressing Venus (KI-Ctrl) or Itga followed by Venus (KI-Itga) contained a transgene of 2721 bp or 6470 bp, respectively, which was flanked by the 5'- and 3'- homology arms. Homology arms of approximately 2 kbp were required to obtain heterozygous recombinant clones. Although two KI-Ctrl cloned cells with accurate chromosomal sequence in non-targeted allele were obtained, all KI-Itga cloned cells had indel mutations. By decreasing the amount of Cas9-expressing plasmid and co-transfecting with the rescue vector containing chromosomal sequences, one KI-Itga5 cloned cells with accurate DNA sequence in non-targeted allele was obtained. </p>
Journal
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- Proceedings for Annual Meeting of The Japanese Pharmacological Society
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Proceedings for Annual Meeting of The Japanese Pharmacological Society 92 (0), 3-P-122-, 2019
Japanese Pharmacological Society
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Keywords
Details 詳細情報について
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- CRID
- 1390846609814535552
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- NII Article ID
- 130007813318
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- ISSN
- 24354953
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- Text Lang
- ja
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- Data Source
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- JaLC
- Crossref
- CiNii Articles
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- Abstract License Flag
- Disallowed