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Lentiviral Vectors to Study the Differential Function of ERK1 and ERK2 MAP Kinases

  • Marzia Indrigo
  • Alessandro Papale
  • Daniel Orellana
  • Riccardo BrambillaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 661)

Abstract

Accumulating evidence indicates that p44ERK1 and p42ERK2 mitogen-activated protein kinases (MAPKs) have distinct quantitative roles in cell signaling. In our recently proposed model of regulation of ERK1 and ERK2, p42 plays a major role in delivering signals from the cell membrane to the nucleus, while p44 acts as a partial agonist of ERK2 toward effectors and downstream activators, thus providing a fine tuning system of the global signaling output. Here, we describe systems to modulate MAPK signaling in vitro and in vivo via lentiviral vector (LV)-mediated gene transfer, using three systems: RNAi with small hairpin RNAs, microRNA-mediated gene knockdown, and expression of signaling-interfering mutants of MEK1. We show, by using proliferation assays in mouse embryo fibroblasts (MEF) and NIH 3T3 cells, that gene knockdown of ERK1 promotes cell proliferation in a manner indistinguishable from a constitutively active MEK1 construct, while ERK2 RNAi causes a significant growth arrest, similar to that observed with the ectopic expression of a dominant negative MEK1 mutant.

Key words

ERK1 ERK2 MEK1 Lentiviral vector RNAi microRNA Gene knockdown Cell proliferation Mouse embryo fibroblast NIH 3T3 

Notes

Acknowledgments

This work was supported by the Michael J Fox Foundation for Parkinson’s Research and the Parkinson’s Disease Society of the UK, as well as by the Italian Ministry of Health (to RB).

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Copyright information

© Springer Science+Business Meida, LLC 2010

Authors and Affiliations

  • Marzia Indrigo
    • 1
  • Alessandro Papale
    • 1
  • Daniel Orellana
    • 1
  • Riccardo Brambilla
    • 1
    Email author
  1. 1.Division of Neuroscience, Institute of Experimental NeurologySan Raffaele Foundation and UniversityMilanoItaly

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