Isolation and Nature of Mouse Embryonic Stem Cell Line Efficiently Producing Female Germ-line Chimeras

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  • UCHIDA Masaki
    National Institute of Animal Industry Division of Animal Science and Technology, Faculty of Agriculture, Okayama University
  • TOKUNAGA Tomoyuki
    National Institute of Animal Industry
  • AIZAWA Shinichi
    The Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center
  • NIWA Koji
    Division of Animal Science and Technology, Faculty of Agriculture, Okayama University
  • IMAI Hiroshi
    National Institute of Animal Industry

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Other Title
  • 雌性化胚性幹細胞の分離とその性質
  • シセイカ ハイセイ カンサイボウ ノ ブンリ ト ソノ セイシツ

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During the course of gene targeting on csk gene, encoding non-receptor protein-tyrosine kinase, a peculiar mutated ES cell line (NZ-2) has been isolated from the XY-type ES cell line (TT-2). When this ES cell line was used for the production of chimeras, a number of normal fertile chimeras were born with female phenotype. The aim of the present experiments was to investigate the reasons why ES cell line originally tended to produce male chimeras resulted in the chimeras with female phenotype.<br>NZ-2 cell line was a mixture consisting of mainly two types of the cells, which had 40 (39%) and 39 (48%) chromosomes. To isolate these two cell populations, NZ-2 was subcloned and isolated FE-1 cell line with 39 chromosomal contents. PCR amplification against sry and zfy genes in FE-1 cells suggested that this cell line was XO-type ES cells in a consequence of lacking Y chromosome. Further PCR analysis was done on the targeted csk gene locus and the insertion of neo gene as the targeting vector on the chromosome. None of these mutations were observed in the FE-1 cell line.<br>FE-1 cells were injected into 8-cell embryos to produce chimeras. Fifty two chimeras, including 37 female mice, were obtained from 312 embryos transferred. Thirteen chimeric mice (62%) from 21 female chimeras proved to be functional germ-line chimeras.<br> Taken together these results, the efficient production of female chimeras from XY-type ES cell line may be caused by naturally occurred loss of Y chromosome during the culture or maintenance of ES cell line. FE-1 cell line isolated in this experiment may be useful as ES cell line producing female germ-line chimeras.

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