An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Regulation of plant gene expression by antisense RNA.

Regulation of gene expression by antisense RNA was first discovered as a naturally-occurring phenomenon in bacteria. Recently natural antisense RNAs have been found in a variety of eukaryotic organisms; their in vivo function is, however, obscure. Deliberate expression of antisense RNA in animal and plant systems has lead to successful down-regulation of specific genes. We will review the current status of antisense gene action in plant systems. The recent discovery that 'sense' genes are able to mimic the action of antisense genes indicates that (anti)sense genes must operate by mechanisms other than RNA-RNA interaction.     

Inhibition of expression of a mouse alpha-globin gene by plasmids that include antisense oligonucleotides.

Plasmid-borne DNAs, corresponding to 68-base oligodeoxynucleotides, synthesized in the antisense or sense configuration and based on the nucleotide sequences of various regions of the mouse alpha-globin mRNA, were introduced with the gene for xanthine-guanine phosphoribosyl transferase from E. coli (Ecogpt) into mouse erythroleukemia (MEL) cells by protoplast fusion. Specific inhibition of the synthesis of alpha-globin was observed only in the cells transformed with the plasmids with antisense 68-mers that corresponded to the cap site as well as the site of initiation of translation of alpha-globin mRNA (Oligo-A); Other plasmids with antisense 68-mers that corresponded to the regions of the exon/intron junctions, the individual exons, or the 3' untranslated region were ineffective. This antisense RNA efficiently reduced the production of alpha-globin to 9-18% of the endogenous level after induction with hexylmethylene-bis-acetoamide (HMBA). Moreover, most of the antisense transformants did not show any decrease in the expression of the c-myc gene at the early phases of differentiation of MEL cells. Thus, we propose a hypothesis that the early decline in levels of c-myc mRNA may be independent of and uncoupled from the program of globin synthesis during the differentiation of MEL cells.     

Estimating the number of plasmids taken up by a eukaryotic cell during transfection and evidence that antisense RNA abolishes gene expression in Physarum polycephalum

We have estimated the statistical distribution of the number of plasmids taken up by individual Jurkat lymphoma cells during electroporation in the presence of two plasmids, one encoding for yellow (EYFP) the other for cyan (ECFP) fluorescent protein. The plasmid concentration at which most of the cells take up only one plasmid or several molecules was determined by statistical analysis. We found that cells behaved slightly heterogeneous in plasmid uptake and describe how the homogeneity of a cell population can be quantified by Poisson statistics in order to identify experimental conditions that yield homogeneously transfection-competent cell populations. The experimental procedure worked out with Jurkat cells was applied to assay the effectiveness of antisense RNA in knocking down gene expression in Physarum polycephalum. Double transfection of flagellates with vectors encoding EYFP and antisense-EYFP revealed for the first time that gene expression can be suppressed by co-expression of antisense RNA in Physarum. Quantitative analysis revealed that one copy of antisense expressing gene per EYFP gene was sufficient to completely suppress formation of the EYFP protein in Physarum.     

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.     

Potential for suppression of E. coli rplj gene expression by antisense RNA

Construction of the recombinant plasmids, containing the antisense sequence of the E. coli rplJ gene under control of lac promoter has shown their effect on the level of the detector rplJ-lacZ gene expression.     

Regulation of gene expression by effectors that bind to RNA

Recent studies have revealed several genetic systems in bacteria that use complex RNA structural elements to monitor regulatory signals and control expression of downstream genes. These include RNA thermosensors, in which an inhibitory structure melts at high temperature, and systems where binding of small RNAs or cellular metabolites modulates the structure of the RNA. The remarkable feature of these systems is the ability of the regulatory RNA elements to specifically sense the regulatory signal, without accessory components, and convey that information to the gene expression machinery via a structural change in the nascent RNA.