title: Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease.
abstract: Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs.
authors.affiliation: Department of Environmental Sciences, School of Life and Environmental Science, Azabu University, Sagamihara, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Hisamatsu
authors.forename: Motoharu
authors.initials: M
authors.lastname: Sakaue
authors.forename: Akiko
authors.initials: A
authors.lastname: Takizawa
authors.forename: Tsubasa
authors.initials: T
authors.lastname: Kato
authors.forename: Maki
authors.initials: M
authors.lastname: Kamoshita
authors.forename: Junya
authors.initials: J
authors.lastname: Ito
authors.forename: Naomi
authors.initials: N
authors.lastname: Kashiwazaki
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus; D004720:Endonucleases / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D005260:Female; D055786:Gene Knockout Techniques / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D016678:Genome / Q000235:genetics*; D051379:Mice; D008845:Microinjections / Q000379:methods; D051381:Rats; D011986:Receptors, Somatotropin / Q000235:genetics*; D013552:Swine / Q000196:embryology* / Q000235:genetics*; D014162:Transfection; D016335:Zinc Fingers; D017388:Zygote Intrafallopian Transfer
chemical_list: D011986:Receptors, Somatotropin; D004720:Endonucleases
keywords: embryo; genome editing; pig; zinc finger nuclease