shRNA Plasmid Expression Vector Construction

RNA interference (RNAi) is a powerful experimental tool in the laboratory, using homologous double-stranded RNA (dsRNA) to induce the silencing of target genes, which can rapidly block gene activity. BOC Sciences offers shRNA plasmid expression vector construction services, allowing customers to use it in their research without worries.

Introduction

shRNA: a small hairpin RNA or short hairpin RNA. It is a sequence of RNA with a tight hairpin turn that is often used for RNA interference to silence the expression of target genes. The shRNA is introduced into the cell using a vector with a U6 promoter that ensures that the shRNA is always expressed; this shRNA-loaded vector can be passed on to daughter cells so that gene silencing can be inherited. shRNA's hairpin structure can be cleaved by the cellular machinery into siRNA, which then binds to the RNA-induced silencing complex (RISC). induced silencing complex (RISC), which binds to and degrades target mRNAs. The shRNA is transcribed by RNA polymerase III and the shRNA product in mammalian cells sometimes induces an interferon response (the cell's self-defense response to a viral attack) that is not encountered in miRNA (miRNA is transcribed by RNA polymerase II, the same polymerase that transcribes mRNA). shRNAs are also used in plants and other systems where the U6 promoter is not required for drive. In plants, the commonly used promoter for enhanced expression is CaMV35S (cauliflower mosaic virus 35S), in which case RNA polymerase II is involved in transcription, initiating RNA interference.

shRNA Vector Construction

shRNA vectors can efficiently express small interfering RNA precursors, carry GFP tags to facilitate expression tracking, and carry screening resistance to facilitate screening of stable transfected strains. In addition, inducible Teton vectors are also available. Under the premise that the transfection efficiency is greater than 80%, the experimental project designs 3 RNA interference sequences for a single gene, which can ensure that at least one can achieve an interference efficiency of more than 70%.

• Experimental Principle

The shRNA sequence is cloned into the silencing vector and transcribed into hairpin RNA, which is cut by Dicer to form a mature siRNA, which in turn leads to mRNA interference of the target gene.

• Service Process
• Design the shRNA sequence, synthesize the sequence and anneal it to generate double-stranded DNA.
• Ligate the DNA to the shRNA expression vector and transform E. coli receptor cells.
• Preparation of plasmids and strains.
• Submit the electronic version of the sequencing result report and carrier information to the client.

• Customer Provided

GeneBank number of the target gene.

• Experiment Provided

The constructed U6 promoter shRNA vector plasmid and corresponding strains.

Applications

• Suitable for experiments requiring observation of the results of transient gene silencing (less than 10 days).
• Suitable for the construction of RNAi stable expression cell lines.
• Suitable for cells with relatively high metabolic levels where low transcription levels may lead to low expression of shRNA plasmids.

Our Advantages

• Effective RNAi product: refers to the knockdown efficiency of the mRNA level of the target gene not less than 70%.
• Short cycle.
• Low price.
• Stable quality.
• Stable cell lines can be constructed.