RNAi Lentivirus Packaging Service

RNAi is one of the most commonly used methods in the study of gene function. BOC Sciences uses lentivirus to integrate the shRNA sequence into the cell genome, and screens out the cells stably transfected with shRNA through the resistance gene (usually neomycin, puromycin, hygromycin) carried by the lentivirus vector, making it a shRNA stably transfects cell lines.

Experimental Principles

RNA interference (RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA, which is highly conserved during evolution. siRNA can form an RNA-induced silencing complex with specific enzymes in the cytoplasm, and siRNA, endonuclease, exonuclease, and helicase are essential components of the complex. Under the action of RISC, the siRNA is unstranded into a single strand, of which the antisense strand specifically recognizes the target mRNA and binds to it. Once bound, the intracellular nucleic acid endonuclease cleaves the target mRNA at the near midpoint of the target mRNA. The cleaved mRNA fragment is degraded by the nucleic acid exonuclease and cannot properly encode into the corresponding protein, resulting in a loss of function.

RNAi, as an important mechanism for post-transcriptional silencing of genes, is widely used in experimental research on gene function, tumor and viral infectious disease treatment. Currently, there are two main techniques: siRNA transfection and construction of shRNA expression vectors.

Lentiviral vectors (LVs) are viral vector systems based on the HIV-1 virus that are highly efficient at introducing target genes (or RNAi) into primary cells or cell lines in animals and humans. The lentiviral vector genome is a positive-strand RNA. After its genome enters the cell, it is reversed into DNA by its own reverse transcriptase in the cytoplasm to form a DNA pre-integration complex. After entering the nucleus, the DNA is integrated into the cell genome. The integrated DNA transcribes mRNA, returns to the cytoplasm, and expresses the target protein; or produces RNAi.

Lentiviral vector-mediated gene expression or RNAi is sustained and stable because the target gene is integrated into the host cell genome and divides as the cell genome divides. In addition, lentiviral vectors can efficiently infect and integrate into non-dividing cells. These characteristics make lentiviral vectors distinctive compared to other viral vectors, such as adenoviral vectors that do not integrate, adeno-associated viral vectors that have low integration rates, and traditional retroviral vectors that only integrate into dividing cells. Numerous studies in the literature have shown that lentiviral vectors mediate long-term expression of target genes in tissues or cells including brain, liver, muscle, retina, bone marrow mesenchymal stem cells, macrophages, etc. The lentiviral vector does not express any HIV-1 protein, has low immunogenicity, no cellular immune response at the injection site and low humoral immune response, and does not interfere with the second injection of the viral vector.

Service Process

1) Construction and purified extraction of lentiviral RNAi vector containing the target gene.

2) Lentiviral interference vector, pGag/Pol, pRev, pVSV-G quad plasmids cotransfected with 293T cells.

3) After culturing for 48-72 hours, collect the culture supernatant.

4) Purification and concentration of viruses (ultra separation and/or ultrafiltration).

5) Titer determination, target gene assay.

6) Transduce the prepared lentiviral liquid into the customer's target cells, screen the mixed stable cell lines, and detect the expression of the target genes.

Customer Provided

1) Plasmids expressing miRNA/shRNA (sequence map provided) or the name and sequence of the target gene to be Knockdowned.

2) The required virus titer and total amount, whether the virus needs to be purified.

3) If it is necessary to detect the expression changes of the target gene, please provide the appropriate antibody if required.

Experiments Provide

1) Provide recombinant lentiviral vector plasmid

2) Provide a lentiviral stock solution with a measured titer that can be directly used for subsequent analysis