title: Production of knockout mice by gene targeting in multipotent germline stem cells.
abstract: Spermatogonial stem cells can convert into embryonic stem (ES) cell-like multipotent germline stem (mGS) cells in vitro and produce germline chimeras by blastocyst microinjection. Although homologous recombination was previously demonstrated in mGS cells, spermatogenesis was not found in chimeras, suggesting that they are not competent for germline modification. Here we conducted detailed analysis of chimeric animals to determine whether mGS cells retain germline potential after genetic manipulation. Spermatozoa that were deficient in the occludin gene could be recovered from animals that were chimeric with mGS cells that underwent homologous recombination. The phenotypes of the occludin knockout (KO) mice were similar to those reported for KO mice produced using ES cells, and the animals showed growth retardation, gastritis and male infertility. Furthermore, we found that heterozygous mGS cells acquire two copies of the G418-resistant genes and become homozygous for the targeted allele by culturing at high concentrations of G418. Cytogenetic analysis showed that the aneuploid mGS cells observed during genetic manipulation were trisomic for chromosome 8 or 11, which is a common chromosomal abnormality in ES cells. Thus, mGS cells can be used to produce KO animals, and this novel method of germline manipulation may prove useful in diverse mammalian species.
authors.affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Takehashi
authors.forename: Mito
authors.initials: M
authors.lastname: Kanatsu-Shinohara
authors.forename: Hiromi
authors.initials: H
authors.lastname: Miki
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Lee
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.forename: Kimiko
authors.initials: K
authors.lastname: Inoue
authors.forename: Narumi
authors.initials: N
authors.lastname: Ogonuki
authors.forename: Shinya
authors.initials: S
authors.lastname: Toyokuni
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.forename: Atsuo
authors.initials: A
authors.lastname: Ogura
authors.forename: Takashi
authors.initials: T
authors.lastname: Shinohara
mesh_terms: D000818:Animals; D002678:Chimera; D005260:Female; D005298:Fertility; D018390:Gene Targeting / Q000379:methods*; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism*; D006720:Homozygote; D007314:Insemination; D008297:Male; D008565:Membrane Proteins / Q000172:deficiency; D051379:Mice; D018345:Mice, Knockout; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D062793:Occludin; D010375:Pedigree; D010641:Phenotype; D013091:Spermatogenesis; D013094:Spermatozoa / Q000378:metabolism; D013737:Testis / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse