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Related Experiment Videos

Knockout mouse production assisted by Blm knockdown.

Mikiko Fukuda1, Mayuko Inoue, Daisuke Muramatsu

  • 1Cellular Memory Laboratory, RIKEN, Saitama 351-0198, Japan.

The Journal of Reproduction and Development
|November 25, 2015
PubMed
Summary

Knocking down Bloom syndrome (BLM) gene expression in embryonic stem cells (ESCs) significantly enhances gene targeting efficiency for producing knockout mice. This improved efficiency facilitates successful germline transmission, aiding in gene function studies.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Developmental Biology

Background:

  • Gene targeting in embryonic stem cells (ESCs) is crucial for creating knockout mice to study gene function in vivo.
  • Homologous recombination (HR) is the established method for gene targeting in ESCs, but efficiency can be locus-dependent and often low.
  • Previous studies showed Bloom syndrome (BLM) knockdown enhances HR in cell lines, but its utility in ESCs for knockout mouse production was unproven.

Purpose of the Study:

  • To investigate if knockdown of the Bloom syndrome gene (BLM) in ESCs can improve gene targeting efficiency for producing knockout mice.
  • To determine if BLM knockdown in ESCs is a viable strategy for generating genetically modified mice with enhanced HR efficiency.

Main Methods:

  • Utilized targeted homologous recombination (HR) for gene targeting in mouse ESCs.

Related Experiment Videos

  • Employed RNA interference (RNAi) to achieve knockdown of the Bloom syndrome gene (BLM) in ESCs.
  • Assessed gene targeting efficiencies across three different gene loci.
  • Evaluated the ability of targeted ESC clones to form chimeras and achieve germline transmission.

    • Main Results:

    • Blm knockdown consistently enhanced HR-mediated gene targeting efficiencies by 2.3- to 4.1-fold across the three examined gene loci.
    • The ESC clones generated from Blm knockdown experiments successfully produced chimeras.
    • Germline transmission was achieved from the targeted ESC clones, confirming the utility of the approach.

    Conclusions:

    • Blm knockdown is an effective strategy to enhance gene targeting efficiency in ESCs.
    • This approach offers a general benefit for efficient production of knockout mice using ESC-based HR.
    • The findings support the use of Blm knockdown to overcome low targeting efficiencies in generating genetically modified mouse models.