shRNA Vector Construction Services

RNAi vector expression can be used to study gene function stably for a long time, and the vector can continue to inhibit the expression of target genes in cells for up to several weeks. Using the resistance gene, the corresponding antibiotics can be used to select stable cell lines that inhibit the expression of the target gene. BOC Sciences offers a comprehensive range of RNAi vector construction services, allowing customers to worry-free and rest assured in their research.

Introduction

RNA interference (RNAi) refers to small double strand RNA (dsRNA) that can efficiently and specifically block the expression of specific genes in vivo and induce mRNA degradation, causing cells to exhibit a specific gene deletion phenotype.

• Molecular Mechanism of RNAi

1) Exogenous genes are randomly integrated into the host cell genome to produce some dsRNAs. The endonuclease Dicer in the cytoplasm cleaves these dsRNAs into multiple short double-stranded RNAs (about 21~25 bp) with specific lengths and structures, i.e. siRNAs.

2) siRNA is unstranded into a sense strand and an antisense strand by the action of RNA helicase, and then the antisense siRNA combines with some enzymes in vivo to form the RNA-induced silencing complex (RISC).

3) RISC binds specifically to the homologous region of mRNA expressed by exogenous genes. RISC functions as a nuclease, cleaving mRNA at the binding site, and the cleaved broken mRNA is then degraded.

4) Amplification of RNAi signal: siRNA can not only guide RISC to cut homologous single-stranded mRNA but also act as a primer to bind to target RNA and synthesize more new dsRNA under the action of RNA-dependent RNA polymerase (RdRP). The newly synthesized dsRNA is then cut by Dicer to generate The newly synthesized dsRNAs are then cleaved by Dicer to produce a large number of secondary siRNAs, which further amplify the effect of RNAi and eventually completely degrade the target mRNA.

• Characteristics of RNAi Action

1) Efficiency

It was found that different concentrations of dsRNA produced the same effect during RNAi.

2) High specificity

RNAi induced by dsRNA only causes the degradation of mRNA homologous to dsRNA, and does not affect the expression of other genes, which have high specificity.

3) ATP-dependent

RNAi is an ATP-dependent process, and exogenous ATP does not promote it.

4) Transmissibility

In plants, RNAi signals can be transmitted between cells via intercellular filaments or vascular tissues. In animals, the diffusion of RNAi signals requires the involvement of specific proteins that form transmembrane channels in the membrane and spread throughout the organism.

5) Heritability

RNAi effects are stably inherited to the offspring, and the phenotype in the offspring is inherited in a Mendelian manner.

• Factors Affecting RNAi Vector Construction

1) Presence or absence of introns

Functional spacer introns produced by splicing during transcription are more effective at mediating gene silencing than nonfunctional introns. The formation of ihpRNA vectors with introns is more than 90% more efficient in silencing than hpRNA.

2) Strength of promoter

The promoter strength has an important effect on the expression level of exogenous genes.

3) Size of the target fragment

Helliwell suggested that a fragment size of 300 bp to 600 bp is more likely to be used to obtain more effective gene silencing.

RNAi Vector Construction Services

• siRNA Synthesis

For transient gene silencing experiments, chemically synthesized siRNAs have the advantages of a simple operation method, easy to obtain high level of transient silencing effect and high specificity. It is suitable for the screening of preliminary RNA interference sequences and transient gene knockdown experiments.

1) Normal siRNA synthesis - HPLC purification with 100% removal of single-stranded RNA.

2) Chemically modified siRNA - 2'-Fluoro or 2'-OMe improves the stability of siRNA in serum and culture medium with longer action time than normal siRNA.

3) Fluorescence-labeled siRNA - Labeled siRNA can be used for flow cytometry, fluorescence microscopy and laser confocal detection, which can facilitate monitoring transfection efficiency and optimizing transfection conditions.

• shRNA Plasmid Expression Vector Construction

The shRNA plasmid is transfected into cells and transcribed within the cells to generate shRNA, which can be used to generate the corresponding siRNA by using the intracellular Dicer enzyme to play the role of RNAi.

• shRNA Viral Expression Vector Construction

Commonly used viral vectors include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, etc. The mechanism is similar to that of plasmid vectors, which take advantage of the relatively high efficiency of viral infection of cells and solve the defect of low transfection efficiency with plasmid vectors.

1) RNAi lentivirus packaging service

2) RNAi adenovirus packaging service

BOC Sciences can provide siRNA/shRNA/miRNA and other sequence design, and provide comprehensive gene expression solutions from siRNA synthesis, shRNA vector construction to related virus packaging, gene silencing function verification, etc. Please feel free to contact us for more details, our scientists will tailor the most reasonable plan for your project. We are happy to serve you.