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A new strategy of gene trapping in ES cells using 3'RACE

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Abstract

“Gene trapping” in embryonic stem (ES) cells is a novel approach to identify a series of genes in mammals concomitant with the production of the corresponding mutant mice. However, this approach is currently unable to identify genes that are not expressed in ES cells. Here we describe a strategy to identify gene trapping clones which is not based on expression of a reporter gene. It uses theneo r gene which lacks a polyadenylation signal and has a splice donor signal. Expression of theneo r gene as fusion transcripts with the 3′ end containing the polyadenylation signal of tagged genes allows the identification of these clones by 3′ rapid amplification of the cDNA end in undifferentiated ES cells, even if the genes are not expressed in ES cells. Amplification was observed in about 25% of G418-resistant clones. Sequence analyses suggested the amplifications represent gene trapping events. The feasibility of this approach was further assessed by analysing one clone, PAT-12, in detail.

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Author notes

  1. Takeshi Yagi

    Present address: Department of Neurobiology and Behavioral Genetics, National Institute for Physiological Sciences, Myodaiji, 444, Okazaki, Japan

  2. Tomoyuki Tokunaga

    Present address: Laboratory of Reproductive Biochemistry, National Institute of Animal Industry, 2 Ikenodai, Kukizaki, Inashiki-gun, 305, Ibaraki, Japan

Authors and Affiliations

  1. Laboratory of Molecular Oncology, Tsukuba Life Science Center, Institute of Physical and Chemical Research, RIKEN, 3-1-1 Koyadai, 305, Tsukuba, Ibaraki, Japan

    Michio Yoshida, Takeshi Yagi, Yasuhide Furuta, Naoki Takeda, Tomoyuki Tokunaga, Yoji Ikawa & Shinichi Aizawa

  2. Department of Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto, University School of Medicine, 2-2-1 Honjo, 860, Kumamoto, Japan

    Michio Yoshida, Yasuhide Furuta, Naoki Takeda & Shinichi Aizawa

  3. Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, 278, Noda, Chiba, Japan

    Michio Yoshida & Joe Chiba

  4. First Laboratory of Biochemistry, Niigata University School of Medicine, 1-757 Asahimachidori, 951, Niigata, Japan

    Kenji Takayanagi & Ryo Kominami

  5. Department of Biochemistry, Tokyo Medical and Dental University School of Medicine, 113, Tokyo, Japan

    Yoji Ikawa

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  1. Michio Yoshida
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Yoshida, M., Yagi, T., Furuta, Y. et al. A new strategy of gene trapping in ES cells using 3'RACE. Transgenic Research 4, 277–287 (1995). https://doi.org/10.1007/BF01969122

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  • DOI: https://doi.org/10.1007/BF01969122

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