PMID- 21370040
OWN - NLM
STAT- MEDLINE
DCOM- 20110608
LR  - 20231127
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 718
DP  - 2011
TI  - iCODA: RNAi-based inducible knock-in system in Trypanosoma brucei.
PG  - 23-37
LID - 10.1007/978-1-61779-018-8_2 [doi]
AB  - 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.
FAU - Ringpis, Gene-Errol
AU  - Ringpis GE
AD  - Department of Microbiology and Molecular Genetics, School of Medicine, University 
      of California, Irvine, CA, USA.
FAU - Lathrop, Richard H
AU  - Lathrop RH
FAU - Aphasizhev, Ruslan
AU  - Aphasizhev R
LA  - eng
GR  - R01 AI091914/AI/NIAID NIH HHS/United States
GR  - R01AI064653/AI/NIAID NIH HHS/United States
GR  - R01CA112560/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (Protozoan Proteins)
RN  - 0 (RNA, Protozoan)
SB  - IM
MH  - Animals
MH  - Cell Line
MH  - Electroporation/methods
MH  - Insecta/cytology
MH  - Mutation
MH  - Plasmids/genetics
MH  - Protozoan Proteins/genetics/*isolation & purification
MH  - *RNA Editing
MH  - *RNA Interference
MH  - RNA, Protozoan/*genetics
MH  - Transfection/methods
MH  - Trypanosoma brucei brucei/*enzymology/*genetics
EDAT- 2011/03/04 06:00
MHDA- 2011/06/09 06:00
CRDT- 2011/03/04 06:00
PHST- 2011/03/04 06:00 [entrez]
PHST- 2011/03/04 06:00 [pubmed]
PHST- 2011/06/09 06:00 [medline]
AID - 10.1007/978-1-61779-018-8_2 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2011;718:23-37. doi: 10.1007/978-1-61779-018-8_2.