Production of Knockout Mice by Random or Targeted Mutagenesis in Spermatogonial Stem Cells

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successfu...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2006-05, Vol.103 (21), p.8018-8023
Main Authors: Kanatsu-Shinohara, Mito, Ikawa, Masahito, Takehashi, Masanori, Ogonuki, Narumi, Miki, Hiromi, Inoue, Kimiko, Kazuki, Yasuhiro, Lee, Jiyoung, Toyokuni, Shinya, Oshimura, Mitsuo, Ogura, Atsuo, Shinohara, Takashi
Format: Article
Language:English
Subjects:
Animal reproduction
Animals
Biological Sciences
Cell lines
Cell transplantation
Embryonic stem cells
Gene targeting
Genetic recombination
Genetic Therapy - methods
Germ cells
Germ Cells - cytology
Germ-Line Mutation
Heterozygote
Male
Mice
Mice, Inbred C57BL
Mice, Inbred DBA
Mice, Knockout
Mutagenesis
Mutation
Neuroglia
Pedigree
Rodents
Somatic cells
Spermatogenesis
Spermatogonia - cytology
Stem cells
Stem Cells - cytology
Testes
Transplantation
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Summary:Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0601139103