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Inducible microRNA-Mediated Knockdown of the Endogenous Human Lamin A/C Gene

  • Ina WeidenfeldEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 815)

Abstract

RNA interference (RNAi) enables the suppression, and hence the functional analysis, of individual genes. The use of the tetracycline (tet)-controlled transcription activation system for RNAi has become a valuable tool for conditional gene inactivation both in vitro and in vivo. Here, the generation of a conditional RNAi cell line for microRNA (miRNA)-mediated downregulation of the endogenous lamin A/C gene is described. A tet-responsive transcription unit, encoding a designed miRNA against human lamin A/C, is directly placed into a predefined genomic site of our previously developed cell line HeLa-EM2-11ht. This chromosomal locus permits the stringent control of miRNA expression, which results in the precise adjustment of lamin A/C protein concentrations. The utilization of this conditional RNAi system for the controlled inactivation of any gene of interest may significantly contribute to the study of gene functions under highly defined conditions.

Key words

Conditional RNA interference Rationally designed microRNA Tet-On system Flp recombinase-mediated cassette exchange Clonal cell lines Lamin A/C Western blotting Immunofluorescence Confocal laser scan microscopy 

Notes

Acknowledgments

I would like to thank the Professors Hermann Bujard and Dirk Görlich as well as Dr. Kai Schönig for their support and encouragement.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Molecular, Cellular, and Developmental BiologyUniversity of Colorado at BoulderBoulderUSA

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