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Production of mouse pups from germline transmission-failed knockout chimeras

Yoshitaka Fujihara1, Kazuhiro Kaseda, Naokazu Inoue

  • 1Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, 565-0871, Osaka, Japan.

Transgenic Research
|July 25, 2012
PubMed

Insights

Researchers successfully produced knockout mice by identifying embryonic stem (ES)-derived germ cells in infertile male chimeras. This method enhances the success rate of gene disruption experiments using green fluorescent protein-tagged ES cells and intracytoplasmic sperm injection.

Area of Science:

  • Reproductive biology
  • Genetics
  • Developmental biology

Background:

  • Gene disruption experiments using chimeras can fail to transmit the gene of interest to offspring.
  • Identifying the contribution of embryonic stem (ES) cells to the germline is crucial for successful gene targeting.

Purpose of the Study:

  • To develop a reliable method for identifying ES-derived germ cells in the testes of chimeric mice.
  • To establish knockout mouse lines from infertile chimeric males.

Main Methods:

  • Generation of chimeric mice using green fluorescent protein (GFP)-tagged ES cells.
  • Identification of ES-derived germ cells in the testis.
  • Production of pups via intracytoplasmic sperm injection (ICSI) from chimeric males.

Main Results:

  • Successfully produced pups from three lines of infertile chimeric male mice.
  • Established knockout mouse lines by combining GFP-tagged ES cells and ICSI.
  • Demonstrated the utility of GFP tagging for identifying ES-derived germ cells in the germline.

Conclusions:

  • Combining GFP-tagged ES cells with ICSI provides a robust strategy for generating knockout mice.
  • This approach mitigates the risk of failure in gene disruption experiments by enabling the identification of functional ES-derived germ cells.

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