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iCODA: RNAi-Based Inducible Knock-In System in Trypanosoma brucei

  • Gene-Errol Ringpis
  • Richard H. Lathrop
  • Ruslan AphasizhevEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 718)

Abstract

In vivo mutational analysis is often required to characterize enzymes that function as subunits of the U-insertion/deletion RNA editing core complex (RECC) in mitochondria of Trypanosoma brucei. The mutations may skew phenotypic manifestation of a dominant negative overexpression if complex association is disrupted. Conditional knockouts and knock-ins of essential mitochondrial genes are time consuming and restricted to the bloodstream form parasites, thus limiting biochemical analysis. We have combined CODA (computationally optimized DNA assembly) technology with RNA interference to develop an iCODA inducible knock-in system for expeditious phenotype assessment and affinity purification of the RECC bearing a mutant subunit. For functional knock-in, the gene region targeted by RNAi is replaced with a synthetic sequence bearing at least one silent mutation per 12 contiguous base pairs. Upon co-expression of the double-stranded RNA targeting the endogenous transcript and modified mRNA in a stable cell line, the endogenous mRNA is destroyed and the cell survives on the RNAi-resistant transcript encoding the same polypeptide. In this chapter, we describe the generation of procyclic (insect) transgenic cell lines, RNAi rescue, complex purification, and validation methods for RNA editing TUTase 2 (RET2). These methods should be readily applicable for any gene in T. brucei.

Key words

Trypanosoma Mitochondria RNA editing RNAi TUTase 

Notes

Acknowledgments

We thank George Cross and Elisabetta Ullu for kind gifts of cell lines and plasmids. This work was supported by the NIH grants RO1AI064653 to RA and R01CA112560 to RHL.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Gene-Errol Ringpis
    • 1
  • Richard H. Lathrop
    • 2
  • Ruslan Aphasizhev
    • 3
    Email author
  1. 1.Department of Microbiology and Molecular Genetics, School of MedicineUniversity of CaliforniaIrvineUSA
  2. 2.Department of Computer Science, School of Information and Computer Sciences, Institute for Genomics and BioinformaticsUniversity of CaliforniaIrvineUSA
  3. 3.Department of Microbiology & Molecular Genetics, School of MedicineUniversity of CaliforniaIrvineUSA

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