A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei

First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.     

Development of a tetracycline controlled gene expression system in the parasitic protozoan Giardia lamblia

Giardia lamblia is a very common intestinal protozoan pathogen of humans. Recent development of gene transfection systems in G. lamblia has allowed constitutive expression of selected genes in the organism. To extend the uses of DNA transfection in G. lamblia, an inducible gene expression system was developed by integrating the bacterial tet operator-repressor elements into an episomal DNA transfection vector. Tetracycline-responsive promoters with insertions of multiple tet operator sequences in the vicinity of a synthetic ran promoter were tested for their inducibility of a luciferase reporter gene expression. Stable cell lines transfected with individual plasmid constructs were established under drug selection. By assaying luciferase activity in transfected cells in response to tetracycline, an inducible promoter with insertion of two tet operators downstream of the adjacent synthetic ran promoter was found to confer a 10-fold inducibility in gene expression with co-expression of the tet-repressor driven by a gdh promoter. To further improve its inducibility, several other synthetic promoter contexts were also tested to increase expression of the tet-repressor gene. An optimal inducibility of 50-fold was obtained when a synthetic alpha-giardin promoter was used. Fine tuning of luciferase expression was achieved by adjusting the concentration of tetracycline and duration of drug exposure. The inducible gene expression system provides us an easy way to manipulate the level of gene expression in G. lamblia in a controllable manner that could not previously be achieved.     

Tetracycline-mediated regulation of gene expression within the human cytomegalovirus genome.

To evaluate the utility of tetracycline gene regulation in the study of human cytomegalovirus gene functions, expression of luciferase under the control of tetracycline-regulatable promoters was studied following transient plasmid transfections and from within recombinant human cytomegalovirus genomes. The tetracycline-regulatable promoter PhCMV*-1 contains sequences from the human cytomegalovirus ie1/ie2 promoter and seven upstream tet operator sites which bind the activator protein tTA only in the absence of tetracycline (Gossen and Bujard (1992). Proc. Natl. Acad. Sci. USA 89, 5547-5551). Two modifications of PhCMV*-1 were also studied: P1129, in which the tet operator sites were reduced from seven to one; and P1125, in which human cytomegalovirus sequences were replaced by adenovirus major late promoter and terminal deoxynucleotidyltransferase initiator sequences. In transient assays, PhCMV*-1 and P1125 exhibited modest differential regulation but were strongly activated by viral infection. P1129 exhibited less viral activation and narrower regulation. In the viral genome, PhCMV*-1 exhibited regulation up to 7-fold during late times of infection, whereas P1125 displayed nearly 100-fold regulation. Regulation of P1125 was fully reversed within 12 to 24 h of adding or removing tetracycline. These results suggest that P1125 may provide sufficient conditional expression to effectively regulate human cytomegalovirus late genes.     

Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi

The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi beta-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.     

Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (approximately 70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.     

Inhibition of hepatitis B virus replication by recombinant small interfering RNAs

RNA interference (RNAi) is a biological phenomenon in which introduction of a small, double-stranded interfering RNAs (siRNAs) into a cell causes a specific degradation of homologous single-stranded RNA. siRNA can be delivered into the cell by different approaches including synthetic RNA, in vitro transcribed RNA and RNA transcribed from polymerase III-based recombinant vectors. As hepatitis B (HB) represents a worldwide health problem, we attempted to develop a fast and easy approach to generation and screening of specific siRNA-targeted HB virus (HBV) genes. Using PCR amplification, specific siRNA expression cassettes (SECs) were developed and used to generate effective siRNAs against HB virus (HBV) replication and gene expression in mammalian cells. After screening, we identified two SECs that expressed siRNAs which efficiently decreased the level of HBV pre-c/c gene expression in transfected Bel-7402 cells by 81.9% and 87.3%, respectively. In addition, the level of HBV DNA was decreased by 83.5% and 85.2% in HepG2 2.2.15 cells, respectively. This study provides (i) a new effective application of RNA interference to study viral gene function and viral replication and (ii) a new tool for the prevention and treatment of human HBV infection.     

Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei

In many eukaryotes, double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10-60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype.