In this article we will demonstrate the expression vector that make antisense gene RNA in laboratory.
Inhibition of gene expression in natural system by antisense RNA led to the development of strategies to artificially regulate genes using antisense RNA. In a single manipulation, antisense RNA complementary to target mRNA can be synthesized in laboratory and may be used to down regulate expression of the target gene.
Experiments with one of the Herpex simplex viral gene thymidine kinase (TK) proved that an artificially antisense RNA gene could be used to regulate the expression of a selected gene in eukaryotes. The antisense TK gene was inserted in sense and antisense orientation between the promoter of TK gene and a viral (SV40) polyadenylation signal. It was later microinjected into animal cell at the ratio of 1:100, sense to antisense, results in lower level of TK gene expression.
It has been demonstrated that expression vector that make antisense gene RNA can be engineered from DNA duplex in the laboratory and delivered into cells. While making antisense gene, plasmid can be cut near a promoter region and DNA duplex (gene) can be spliced into the plasmid to create an expression vector. Efficiency of expression vector in cells can be confirmed by initiating transcription that will make copies of mRNA.
If the added DNA is then cut out of the expression vector it can reinsert itself into the ring with opposite orientation. Thus, during transcription expression vector contains reinserted DNA in antisense orientation will make antisense RNA. Once expression vector is introduced into cells that will make copies of antisense RNA after transcription. This antisense RNA will then binds specifically with target mRNA in a cell and prevent its translation.
Apart from rely on expression vectors strands of antisense RNA can be synthesizing in the laboratory and injected directly into cells in a specialized process known as microinjection (Fig. 22.2). This approach can exploit possibilities of directly introducing specific antisense RNA into individual cells.
This system is highly viable for certain biological system that lacks methods for stable transformation. In 1985, Douglas A. Melton of Harvard University reported that direct microinjection of antisense β-globin RNA into larger frog oocytes could inhibit the translation of corresponding sense (mRNA) β-globin RNA injected previously.
When RNA was extracted and analysed, protected duplex RNA was found only in cells containing both sense globin mRNA and antisense RNA indicating that hybridization takes place occur in vivo and its hybridization was found to be a time-bound process. In addition, inhibition of chloromphenicol acetyl transferase (CAT) has been accomplished by using specific antisense gene.
Similar results were obtained by RNA microinjection experiments. The mRNA for these enzymes were injected into oocytes and were shown to be inhibited by corresponding antisense RNA, who were injected five times earlier. In the subsequent experiments on the production of large quantities of antisense RNA, Barbara J.
Word of California Institute of Technology employed cultured cells as it occasionally amplifies genes by making multiple copies of them. They construct antisense expression vector containing gene for essential enzyme and inserted vector into cells. Selection of cells in subsequent generation that had amplified the enzyme gene was utilised to produce large quantity of antisense RNA.