Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells
- Mito Kanatsu-Shinohara*,†,‡,§,
- Masahito Ikawa‡,¶,
- Masanori Takehashi*,‡,
- Narumi Ogonuki‖,
- Hiromi Miki‖,
- Kimiko Inoue‖,
- Yasuhiro Kazuki**,
- Jiyoung Lee*,
- Shinya Toyokuni††,
- Mitsuo Oshimura**,
- Atsuo Ogura‖, and
- Takashi Shinohara*,§
- *Department of Molecular Genetics,
- †Horizontal Medical Research Organization, and
- ††Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan;
- ¶Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan;
- ‖Bioresource Center, The Institute of Physical and Chemical Research (RIKEN), Ibaraki 305-0074, Japan; and
- **Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan
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Edited by Ryuzo Yanagimachi, University of Hawaii, Honolulu, HI, and approved March 27, 2006
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↵ ‡M.K.-S., M.I., and M.T. contributed equally to this work. (received for review February 11, 2006)
Abstract
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.
Footnotes
- §To whom correspondence may be addressed at: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. E-mail: tshinoha{at}virus.kyoto-u.ac.jp or mshinoha{at}virus.kyoto-u.ac.jp
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See Commentary on page 7939.
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Author contributions: M.K.-S. and T.S. designed research; M.K.-S., M.I., M.T., N.O., H.M., K.I., Y.K., J.L., A.O., and T.S. performed research; M.I. contributed new reagents/analytic tools; M.K.-S., M.I., S.T., M.O., A.O., and T.S. analyzed data; and M.K.-S. and T.S. wrote the paper.
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Conflict of interest statement: No conflicts declared.
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This paper was submitted directly (Track II) to the PNAS office.
- Abbreviations:
- EGFP,
- enhanced GFP;
- ES,
- embryonic stem;
- GS,
- germ-line stem;
- neo,
- neomycin.
Abbreviations:
- © 2006 by The National Academy of Sciences of the USA
