title: Disruption of exogenous eGFP gene using RNA-guided endonuclease in bovine transgenic somatic cells.
abstract: 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.
authors.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.
authors.forename: WooJae
authors.initials: W
authors.lastname: Choi
authors.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.
authors.forename: SooYoung
authors.initials: S
authors.lastname: Yum
authors.affiliation: Embryo Research Center in Seoul Milk Coop.,Gyeonggi-do,Republic of Korea.
authors.forename: SongJeon
authors.initials: S
authors.lastname: Lee
authors.affiliation: Embryo Research Center in Seoul Milk Coop.,Gyeonggi-do,Republic of Korea.
authors.forename: WonWu
authors.initials: W
authors.lastname: Lee
authors.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.
authors.forename: JiHyun
authors.initials: J
authors.lastname: Lee
authors.affiliation: Toolgen,INC.,Seoul,Republic of Korea.
authors.forename: SeokJoong
authors.initials: S
authors.lastname: Kim
authors.affiliation: Laboratory Animal Research Center,Samsung Biomedical Research Institute,Gyeonggi-do,Republic of Korea.
authors.forename: OkJae
authors.initials: O
authors.lastname: Koo
authors.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.
authors.forename: ByeongChun
authors.initials: B
authors.lastname: Lee
authors.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.
authors.forename: Goo
authors.initials: G
authors.lastname: Jang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000502:physiology; D064113:CRISPR-Cas Systems; D002417:Cattle; D002478:Cells, Cultured; D004720:Endonucleases / Q000235:genetics; D005260:Female; D005347:Fibroblasts; D055786:Gene Knockout Techniques / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004720:Endonucleases
keywords: CRISPR-Cas9; PiggyBac; Transgenic cattle; eGFP disruption