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Disruption of exogenous eGFP gene using RNA-guided endonuclease in bovine transgenic somatic cells

Published online by Cambridge University Press:  26 November 2014

WooJae Choi ,
SooYoung Yum ,
SongJeon Lee ,
WonWu Lee ,
JiHyun Lee ,
SeokJoong Kim ,
OkJae Koo ,
ByeongChun Lee  and
Goo Jang
WooJae Choi
Affiliation:
Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Republic of Korea.
SooYoung Yum
Affiliation:
Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Republic of Korea.
SongJeon Lee
Affiliation:
Embryo Research Center in Seoul Milk Coop., Gyeonggi-do, Republic of Korea.
WonWu Lee
Affiliation:
Embryo Research Center in Seoul Milk Coop., Gyeonggi-do, Republic of Korea.
JiHyun Lee
Affiliation:
Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Republic of Korea.
SeokJoong Kim
Affiliation:
Toolgen, INC., Seoul, Republic of Korea.
OkJae Koo
Affiliation:
Laboratory Animal Research Center, Samsung Biomedical Research Institute, Gyeonggi-do, Republic of Korea.
ByeongChun Lee
Affiliation:
Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, Republic of Korea.
Goo Jang*
Affiliation:
Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151–742Korea. Emergence Center for Food-Medicine Personalized Therapy System, Advanced Institutes of Convergence Technology, Seoul National University, Gyeonggi-do, Republic of Korea.
*
All correspondence to: Goo Jang. Laboratory of Theriogenology and Biotechnology, Department of Veterinary Clinical Science, College of Veterinary Medicine and the Research Institute of Veterinary Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151–742Korea. Tel: +82 2 880 1280. Fax: +82 873 1269. e-mail: snujang@snu.ac.kr
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Summary

Genome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis. CRISPR/Cas9 plasmids specifically targeting the eGFP gene were transfected into the eGFP fibroblasts by electroporation. After 10 days of culture, more than 40% of the cells had lost eGFP expression in fluorescence activated cell sorting (FACS) analysis. Targeted sequences of the transfected cells were analyzed, and various small indel mutations (6–203 bp deletions) in the target sequence were found. The fibroblasts mutated with the CRISPR/Cas9 system were applied for somatic cell nuclear transfer, and the reconstructed embryos were successfully developed into the blastocyst stage. In conclusion, the CRISPR/Cas9 system was successfully utilized in bovine cells and cloned embryos. This will be a useful technique to develop livestock transgenesis for agricultural science.

Keywords

CRISPR-Cas9eGFP disruptionPiggyBacTransgenic cattle
Type
Research Article
Information
Zygote , Volume 23 , Issue 6 , December 2015 , pp. 916 - 923
Copyright
Copyright © Cambridge University Press 2014 

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