[Targeted exogenous EGFP gene editing in caprine fetus fibroblasts by zinc-finger nucleases].
Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.
Yuguo
Y
Yuan
Baoli
B
Yu
Shaozheng
S
Song
Feng
F
Zhou
Liqing
L
Zhang
Yingying
Y
Gu
Minghui
M
Yu
Yong
Y
Cheng
D000818:Animals; D001483:Base Sequence; D019976:Cloning, Organism; D004586:Electrophoresis; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D005333:Fetus; D005347:Fibroblasts / Q000378:metabolism; D055786:Gene Knockout Techniques; D018390:Gene Targeting / Q000379:methods*; D006041:Goats; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016335:Zinc Fingers
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004720:Endonucleases