Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method
-
- Ezaki Ryo
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University
-
- Ichikawa Kennosuke
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University
-
- Matsuzaki Mei
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University
-
- Horiuchi Hiroyuki
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University
Bibliographic Information
- Other Title
-
- Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homologue Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method
Search this article
Abstract
<p>In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.</p>
Journal
-
- The Journal of Poultry Science
-
The Journal of Poultry Science 59 (2), 182-190, 2022
Japan Poultry Science Association
- Tweet
Keywords
Details
-
- CRID
- 1390291932664103296
-
- NII Article ID
- 130008093959
-
- NII Book ID
- AA11564513
-
- ISSN
- 13490486
- 13467395
-
- NDL BIB ID
- 032109900
-
- Text Lang
- en
-
- Data Source
-
- JaLC
- NDL
- Crossref
- CiNii Articles
- KAKEN
-
- Abstract License Flag
- Disallowed