title: Targeted gene deletion of miRNAs in mice by TALEN system.
abstract: Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.
authors.affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Takada
authors.forename: Tempei
authors.initials: T
authors.lastname: Sato
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Ito
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamashita
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kato
authors.forename: Miyuri
authors.initials: M
authors.lastname: Kawasumi
authors.forename: Masami
authors.initials: M
authors.lastname: Kanai-Azuma
authors.forename: Arisa
authors.initials: A
authors.lastname: Igarashi
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kato
authors.forename: Moe
authors.initials: M
authors.lastname: Tamano
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Asahara
mesh_terms: D000818:Animals; D001483:Base Sequence; D021901:DNA, Intergenic / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004622:Embryo, Mammalian; D005260:Female; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering; D051379:Mice; D008813:Mice, Inbred ICR; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D021901:DNA, Intergenic; D004268:DNA-Binding Proteins; C569259:MIRN10 microRNA, mouse; D035683:MicroRNAs; D011993:Recombinant Fusion Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific