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This observation demonstrates the existence of a duplication of the ace-1 gene that resulted in one susceptible copy and one resistant copy, both of which are on the same chromosome.
The short cilia phenotype is rescued by a shRNA resistant copy of GLOD4 showing that the short cilia phenotype is because of reduced levels of GLOD4 and not from off target effects.
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The shRNA resistant copies of these genes are functional because they rescue the ciliary phenotypes and these genes are clearly involved in ciliogenesis because upon knockdown the cells have fewer cilia and/or short cilia.
All of these phenotypes could be rescued by a shRNA-resistant copy of SPATA4.
This strategy, elaborated using the TbPFR2 gene as a model system, involved the silencing of the TbPFR2 target via its UTRs and the expression of a RNAi-resistant copy of the targetted gene.
In order to address that specific problem, we elaborated a method that involves RNAi experiments on trypanosomes that were engineered to possess an extra RNAi-resistant copy of the targeted gene, leading to functional complementation.
In this report, we demonstrated that RNAi-mediated silencing of a gene by targeting its UTRs is useful in studies where the loss of function resulting from this silencing must be complemented with the expression of an RNAi-resistant copy of the silenced gene, in order to demonstrate that the phenotype is indeed due to silencing of that gene, and not to inactivation of another one.
All of our ciliary phenotypes were rescued by shRNA-resistant copies of our genes.
This region is most resistant to copy number gain.
The genomes of the cladosporin-sensitive parental clone and cladosporin-resistant clones were compared using a microarray that has the capacity to reveal most genomic changes that are acquired by the resistant strains, including copy number variants, deletions, small insertion/deletion events, and single-nucleotide polymorphisms (Dharia et al., 2009).
Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications.
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