iCODA: RNAi-Based Inducible Knock-In System in Trypanosoma brucei
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.
- 差异基因研究技术及其应用
- Lentivirus-Mediated RNA Interference in Mammalian Neurons
- Methods for MicroRNA Microarray Profiling
- An Overview of C. elegans Biology
- Genome-Wide Analysis of Transcription Factor-Binding Sites in Skeletal Muscle Cells Using ChIP-Seq
- Wide Host-Range Cloning for Functional Metagenomics
- Construction of a Normalized cDNA Library by mRN-cDNA Hybridization and Subtraction
- Development and Application of a Dual-Purpose Nanoparticle Platform for Delivery and Imaging of siRNA in Tumors
- DNA Extraction from Plasma and Serum
- Generation of Gene-Specific Mutated Rats Using Zinc-Finger Nucleases