Construction of RNAi expression vector

Recent studies have shown that some short-segment double-stranded RNA can efficiently and specifically block specific gene expression in the body by causing mRNA degradation of specific genes, and induce cells to exhibit a phenotype of specific gene deletion, called RNA interference ( RNA interference, RNAi). siRNA (small interfering RNAs) are short-segment double-stranded RNA molecules that can target specific mRNAs with homologous complements and degrade specific mRNAs. The discovery of RNAi is of epoch-making significance. It reveals the mechanism of intracellular gene silencing, and it is also a powerful tool for gene function analysis in the post-genome era, which greatly promotes the process of revealing the mysteries of life. Now more and more researchers are using RNAi to express gene expression in graduate students. .RNAi technology can be widely used to include functional genomics, drug target screening, cell signaling pathway analysis, disease treatment and more.

The five commonly used methods for preparing siRNAs to date include:

·Chemical synthesis

·In vitro transcription

Long-segment dsRNAs are degraded by RNase III (eg Dicer, E. coli, RNase III)

· siRNA expression vector or viral vector expresses siRNAs in cells

· PCR-prepared siRNA expression cassette is expressed in cells

Obtaining high-purity siRNA products is the first step in the experiment, and the efficiency of transfection is a very critical factor.

First, the basic concept

RNAi: (RNA interference) RNA interference

Endogenous or exogenous double-stranded RNA (dsRNA) mediated by the ability to induce silencing or inhibition of specific gene expression homologous to its sequence in a cell, inducing cells to exhibit a phenotype of a specific gene deletion, known as RNA interference It is also an important protective mechanism against external infection in the body.

siRNA :(small interfering RNAs) small interfering RNA

A 19-25 nt short-sequence double-stranded RNA molecule cleavage by long dsRNA is capable of degrading specific mRNA, a key effector molecule of RNAi, with a homologous complementary sequence of RNA.

shRNAs: (small hairpin RNA ) small hairpin RNA

It is a non-coding small RNA molecule designed to form a hairpin structure. The shRNA needs to be introduced into the cell through the vector, and then the siRNA is obtained by intracellular digestion mechanism to finally exert RNA interference.

Dicer: belongs to the RNaseIII family and is a specific endonuclease of dsRNA

RISC: (RNA-inducing silencing complex) RNA-induced silencing complex with endonuclease, exonuclease and helicase activity

Second, the mechanism

It is generally believed that co-suppression, gene suppression and RNAi are likely to have the same molecular mechanism, specifically degrading target mRNA through dsRNA-mediated degradation of the corresponding gene expression. RNAi, co-suppression, quelling are all PTGS! It has been preliminarily elucidated that the dsRNA-mediated homologous target mRNA degradation process is mainly divided into two steps.

The first step (initial phase) is that small ds RNA is cleaved by ARN in the presence of ARNase III-like specific nuclease to form 21~23 nt of small interfering RNA (siRNA) consisting of sense and antisense strands.

The second step (effect phase) is that the siRNA is decomposed into a single strand by RNA helicase with the participation of ATP, and the antisense strand is used to guide the formation of RNA-induced silencing complex (RISC).

The RNAi pathway is mainly present in the cytoplasm, but the subcellular location of siRNA production and degradation of target mRNA is not clear. Exogenous (injected or fed) dsRNA and viral dsRNA may directly enter the RNAi pathway in the cytoplasm, only in RNA viruses replicated in cytoplasm can be inhibited by dsRNA-mediated silencing mechanisms. Exogenous dsRNA can also result in reduced homologous early RNA transcripts in the nucleus.

In many organisms, the inverted repeat transgene sequence is transcribed into the hairpin dsRNA in the nucleus, which in turn mediates RNAi. This dsRNA may need to be transferred to the cytoplasm to effectively silence the homologous target mRNA.

Amplification effect mechanism of RNAi

siRNA can not only guide RISC to cleave target RNA, but also can be used as a primer to synthesize new dsRNA with target mRNA as template under the action of RNA-dependent RNA polymerase (RdRP).

The newly synthesized long-chain dsRNA can also be cleaved and degraded by RNaseIII-like nuclease to generate a large number of secondary siRNAs. Secondary siRNA can enter the synthesis-cleavage cycle process to further amplify RNAi action. This synthesis-cutting cycle It is called random degradative PCR.

Third, the construction of RNAi expression vector

1. Determination of the target gene

(1) Searching for documents to obtain experimentally valid target sequences (check)

(2), http://

(3), http:// or http://scholar.google.com

2. Design siRNA target sequences

The siRNA sequences need to be designed separately before preparation of siRNA. The most effective siRNAs for mammalian cells are double-stranded RNAs with 21-23 bases and 3' ends with two prominent bases; It is more effective that the sequence specificity of long fragment dsRNA.siRNA is very stringent, and a base mismatch with the target mRNA will significantly impair the effect of gene silencing.

(1) Select siRNA target site:

Starting from the transcription of the AUG start codon, search for the downstream AA sequence and record 19 nucleotides adjacent to each AA 3' end as candidate siRNA target sites. Studies have shown that the GC content is 30%-50%. siRNAs on the left and right are more effective than those with higher GC content. Tuschl et al. suggested not to target non-translated regions (UTRs) at the 5' and 3' ends when designing siRNAs because of the abundant regulatory protein binding in these regions. Regions, and these UTR binding proteins or translation initiation complexes may affect the binding of the siRNP endonuclease complex to mRNA to affect siRNA.

(2), sequence homology analysis:

Compare potential sequences to the corresponding genomic databases (human, mouse, rat, etc.) and exclude those sequences that are homologous to other coding sequences/ESTs. For example, use BLAST ( /BLAST/) Select the appropriate target sequence for synthesis. Not all eligible siRNAs are equally effective. The reason is not clear. It may be the result of positional effect. Therefore, for a target gene, 3-5 targets are generally selected. Site to design siRNA.

Generally speaking, each target sequence is designed with 3-4 pairs of siRNAs, and the most effective selection is for subsequent studies. (3) Design negative control:

A complete siRNA experiment should have a negative control. The siRNA as a negative control should have the same composition as the selected siRNA sequence, but has no obvious homology with the mRNA. The usual practice is to hit the base sequence in the selected siRNA. Chaos. Of course, it is also necessary to ensure that it has no homology with other genes.

3. Selection of RNAi expression vector

Both chemical synthesis and in vitro transcription methods are introduced into cells after siRNA is obtained in vitro, but these two methods mainly have two disadvantages that cannot be overcome: the siRNA is easily degraded after entering the cell; the RNAi effect of entering the cell siRNA in the cell continues. In view of this situation, plasmid and viral vector-mediated expression of siRNA in vivo has emerged. The basic idea of ​​this method is to clone the DNA double-stranded template sequence corresponding to siRNA into the vector of RNA polymerase III promoter. In this way, the desired siRNA molecule can be expressed in vivo. The overall advantage of this method is that it does not require direct manipulation of RNA, and can achieve a long time of gene silencing effect.

The plasmid-expressing siRNAs mostly use the Pol III promoter to initiate the sequence encoding the shRNA (small hairpin RNA). The reason for selecting the Pol III promoter is that the promoter always starts to transcribe and synthesize RNA at a fixed distance from the promoter. It is very accurate to terminate by 4-5 consecutive U. When the plasmid carrying the Pol III promoter and shRNA template sequence is transfected into mammalian cells, the plasmid expressing siRNA can indeed down-regulate the expression of specific genes. It can inhibit foreign genes and endogenous genes. The advantage of using plasmids is that the siRNA vector can inhibit the expression of the target gene for a longer period of time through the selection marker of the siRNA expression plasmid. Of course, the plasmid can be replicated and amplified. This can significantly reduce the cost of preparing siRNA compared to other synthetic methods.

In addition, siRNA expression vectors with antibiotic markers can be used for long-term inhibition studies. By resistance-assisted screening, the plasmid can continuously inhibit the expression of target genes in cells for several weeks or longer. At the same time, RNAi-Ready expression vectors can also be reversed. Integration of viral and adenoviral expression systems (BD Knockout RNAi Systems) greatly enhances the infectivity of siRNA expression vectors to host cells, completely overcomes the barriers of low transfection efficiency of certain cells, and achieves transient expression and stabilization of siRNAs in mammalian cells. The ideal tool for expression.

4, synthetic template

The two single strands of the DNA template encoding the shRNA are synthesized, and the RNA polyIII polymerase transcriptional stop site is followed by the template strand, and the BamH I and Hind III restriction sites are designed at both ends to clone into the siRNA vector multiple cloning site. Between the BamH I and Hind III restriction sites.

95 ° C, 5 min, slow annealing, DNA single stranded DNA shRNA DNA double stranded template

5, connection and conversion

The basic steps:

(1) Thaw 100 μl of competent cells on ice.

(2) Add 5 μl of the ligation product to the competent cells and gently rotate several times to mix the contents. Place on ice for 30 minutes.

(3) Put the tube into a water bath pre-warmed to 42 ° C, heat shock for 90 seconds. Quickly transfer the tube to the ice bath, and let the cells cool for 1-2 minutes.

(4) Add 700 μl of LB medium to each tube and incubate for 1 hour at 37 ° C for resuscitation.

(5) Centrifuge at 4,000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells with the remaining 100 μl of medium and apply to the surface of the resistant LB agar plate. Note: The amount of cells should be based on the efficiency of the ligation and the competent cells. Efficiency adjustment.

(6) Place the plate at room temperature until the liquid is absorbed.

(7) Invert the plate and incubate at 37 ° C. Colonies may appear after 12 to 16 hours.

6, PCR identification and sequencing identification

PCR primers were designed on both sides of the DNA double-stranded template inserted into the shRNA, and the amplified fragment was between 100-200 bp.

7, characteristics

The advantage of siRNA expression vectors is that this is the only method that can be used for long-term studies in many ways - vectors with antibiotic markers can continue to inhibit target gene expression in cells for weeks or longer. Even for transfection bands Transient screening of cells with a marker plasmid also facilitates the enrichment of plasmid-bearing cells. This can also help solve some of the problems of poorly transfected cells due to low transfection efficiency.

The resistance marker on the vector helps to rapidly screen out positive clones, and can continue to inhibit the expression of target genes in the cells for several weeks or longer, allowing for longer-term studies.

Fourth, the prospect of RNAi

1. New tools for studying gene function

Studies have shown that RNAi can inactivate or reduce the expression of specific genes in mammals, produce a variety of phenotypes, and the time to inhibit gene expression can be controlled at any stage of development, producing a similar knock-out effect. And the complete genome sequence of Drosophila has been tested, and a large number of new genes with unknown functions are found. RNAi will greatly promote the study of the function of these new genes. Compared with traditional gene knockout technology, this technology has less investment and shorter cycle. The advantages of simple operation and so on, and the recent reports that RNAi has been successfully used to construct transgenic animal models are increasing, indicating that RNAi will become an indispensable tool for studying gene function.

2. New ways to study signal transduction pathways

The combination of traditional deletion mutation technology and RNAi technology can easily determine the upstream and downstream relationship of different genes in complex signaling pathways. Clemensy et al. applied RNAi to study insulin signaling pathways in Drosophila cell lines, and obtained insulin with known insulin. The relationship between DSH3PX1 and DACK was analyzed based on the complete agreement of the information transmission pathway. It was confirmed that DACK is an upstream kinase located in DSH3PX1 phosphorylation. RNAi technology is simpler, faster and more reproducible than traditional transfection experiments. Overcome the turn

In the dyeing experiment, the specific efficiency of recombinant protein aggregation and transfection is not high, so it is believed that RNAi technology may become a new way to study cell signaling pathway.

3. New strategies for gene therapy

RNAi has the functions of resisting viral invasion, inhibiting transposon activity, and preventing excessive proliferation of selfish gene sequences. Therefore, RNAi can be used to produce antiviral plants and animals, and dsRNAs corresponding to highly homologous segments in different viral transcription sequences can be utilized. Resist against multiple viruses.

Tumors are the result of gene network regulation of multiple gene interactions. The blocking of a single oncogene induced by traditional techniques cannot completely inhibit or reverse the growth of tumors, and RNAi can utilize multiple genes of the same gene family to have a homology. The highly conserved sequence is designed to target dsRNA molecules of this segment sequence. Injecting only one dsRNA can produce simultaneous deletion of multiple genes, or multiple dsRNAs can be injected simultaneously to unequate multiple sequences. The genes are simultaneously removed.

Although the current application of RNAi technology in mammals is still in the exploratory stage, its successful application in vertebrates such as zebrafish and mice indicates that RNAi will become an important component of gene therapy, artificially synthesized dsRNA oligomeric drugs. Development will likely become an emerging industry with great development prospects.