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Generation of Mouse Embryos with Small Hairpin RNA-Mediated Knockdown of Gene Expression

  • David A. F. Loebel
  • Tania Radziewic
  • Melinda Power
  • Joshua B. Studdert
  • Patrick P. L. Tam
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1092)

Abstract

We are using knockdown of gene expression in mouse embryos by constitutive expression of small hairpin (sh)RNAs as a means of observing loss-of-function phenotypes more rapidly than gene targeting. Plasmid constructs that direct shRNA expression via an RNA pol III promoter are introduced into embryonic stem (ES) cells by electroporation and drug selection. Clones are propagated and the degree of knockdown assessed by quantitative protein or RNA methods. Selected ES cell clones are used to generate embryos by tetraploid complementation. Blastomeres of two cell embryos are electrofused to generate tetraploid embryos. Chimeric embryos are produced by injection of ES cells into blastocysts or aggregation with morulae. In these embryos, the tetraploid cells become excluded from the fetal tissues, resulting in ES cell-derived embryos harboring the shRNA knockdown construct. Embryos can be collected and their phenotype assessed by appropriate means.

Key words

Mouse embryo Tetraploid complementation shRNA RNA interference Knockdown Embryonic stem cell Blastocyst injection Aggregation 

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • David A. F. Loebel
    • 1
  • Tania Radziewic
    • 1
  • Melinda Power
    • 1
  • Joshua B. Studdert
    • 1
  • Patrick P. L. Tam
    • 1
  1. 1.Children’s Medical Research Institute, Sydney Medical SchoolThe University of SydneySydneyAustralia

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