title: Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases.
abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.
authors.affiliation: Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Watanabe
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Umeyama
authors.forename: Hitomi
authors.initials: H
authors.lastname: Matsunari
authors.forename: Shuko
authors.initials: S
authors.lastname: Takayanagi
authors.forename: Erika
authors.initials: E
authors.lastname: Haruyama
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Nakano
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Yuka
authors.initials: Y
authors.lastname: Ikezawa
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D053837:DNA Cleavage; D004252:DNA Mutational Analysis; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D018274:Electroporation; D055786:Gene Knockout Techniques / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D034421:Sus scrofa / Q000235:genetics*; D016335:Zinc Fingers
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases