Affiliation:
1. Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, USA
Abstract
ABSTRACT
Conditional gene silencing by RNA interference in
Trypanosoma brucei
can be inconclusive if knockdowns are inefficient or have off-target effects. To enable efficient, specific silencing of single-copy genes in mammalian-infective, bloodstream form trypanosomes, we developed a system that targets the heterologous and functional
Trypanosoma cruzi
U2AF35
3′ untranslated region (UTR) (Tc3) or, alternatively, the sequence of the PTP tag, which can be fused to any mRNA of interest. Two cell lines were created, single-marker Tc3 (smTc3) and smPTP, which conditionally express Tc3 and PTP double-stranded RNA (dsRNA), respectively. The system depends on manipulating both alleles of the gene of interest so that cells exclusively express the target mRNA as a fusion to one of these heterologous sequences. We generated allele integration vectors in which the C-terminal part of a gene's coding sequence can be fused to either heterologous sequence in a single cloning step. We first tested this system with
CITFA7
, which encodes a well-characterized subunit of the class I transcription factor A (CITFA), an essential factor for transcription initiation by RNA polymerase I. Targeting either Tc3 or PTP fused to the
CITFA7
mRNA resulted in gene knockdowns that were as efficient and specific as targeting the endogenous
CITFA7
mRNA. Moreover, application of this system to
CITFA1
, which could not be silenced by established methods, demonstrated that the gene encodes an essential CITFA subunit that mediates binding of the transcription factor complex to RNA polymerase I promoters.
Publisher
American Society for Microbiology
Subject
Molecular Biology,General Medicine,Microbiology
Cited by
7 articles.
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