Construction of siRNA expression vectors

Summary

The construction of siRNA expression vectors can be applied to: (1) as a powerful tool for gene function analysis in the post-genomic era; (2) genomics and cell signaling pathway analysis; (3) drug target screening and disease treatment.

Operation method

Construction of siRNA expression vector

Principle

Most siRNA expression vectors rely on one of three RNA polymerase III promoters (pol III) to manipulate the expression of a small hairpin RNA (shRNA) in mammalian cells. These three types of promoters include the familiar human and murine U6 promoters and the human H1 promoter. The RNA pol III promoter was used because it allows for the expression of more small-molecule RNAs in mammalian cells and because it terminates transcription by the addition of a string (three to six) of U's. To use these vectors, 2 single strands of DNA encoding short hairpin RNA sequences are ordered, annealed, and cloned downstream of the pol III promoter in the appropriate vector. Because of the cloning involved, this process can take weeks or even months, and sequencing is also required to ensure that the cloned sequence is correct.

Materials and Instruments

Target gene
LB medium
Centrifuge Tubes Centrifuge Water Bath Vortex Oscillator

Move

I. Identification of target genes1. Search the literature to obtain target sequences with experimentally proven validity (check).2. NCBI accessed: http://www.ncbi.nlm.nih.gov/.3. Search engine assistance: http://www.google.cn/ or http://scholar.google.com.II. Designing siRNA target sequencesBefore preparing siRNA, we need to design the siRNA sequence individually. It is found that for mammalian cells, the most effective siRNAs are double-stranded RNAs of 21-23 bases in size with two prominent bases at the 3' end, while for non-mammalian animals, the more effective ones are long fragment dsRNAs. siRNA sequence specificity requirements are very strict, and a base mismatch with the target mRNA will significantly weaken the effect of gene silencing.1. Selection of siRNA target sitesStarting from the start codon of transcribed AUG, we searched the downstream AA sequences and recorded the 19 nucleotides adjacent to the 3' end of each AA as candidate siRNA target sites. Some studies have shown that siRNAs with GC content around 30%-50% are more effective than those with high GC content. Tuschl et al. suggested not to target untranslated regions (UTRs) at the 5' and 3' ends of the siRNAs when designing the siRNAs, because these regions are rich in binding regions of regulatory proteins and these UTRs bind proteins or translation initiation complexes. The reason is that these regions are rich in regulatory protein binding regions, and these UTR-binding proteins or translation initiation complexes may affect the binding of mRNA by the siRNP nucleic acid endonuclease complex, thus affecting the effect of the siRNA.2. Sequence homology analysisCompare the potential sequences with the corresponding genomic databases (human or mouse, rat, etc.) and exclude those sequences that are homologous to other coding sequences/ESTs. For example, use BLAST (www.ncbi.nlm.nih.gov/BLAST/) to select suitable target sequences for synthesis. Not all eligible siRNAs are equally effective, the reason for this is unclear and may be a result of positional effects, so for a single target gene, 3-5 target sites are generally selected for siRNA design.In general, 3-4 pairs of siRNAs are designed for each target sequence, and the most effective ones are selected for subsequent studies.

3. Design of negative controlsA complete siRNA experiment should have a negative control. The siRNA used as the negative control should have the same composition as the selected siRNA sequence, but no obvious homology with the mRNA. It is common practice to scramble the base sequences in the selected siRNA. Of course, it is equally important to ensure that it has no homology with other genes.Selection of expression vectors1. Chemical synthesis and in vitro transcription methods are in vitro to get siRNA and then imported into the cell, but these two methods mainly have two insurmountable shortcomings: siRNA into the cell is easy to be degraded; into the cell siRNA in the cell RNAi effect lasts a short time. In response to this situation, plasmid, viral vector-mediated in vivo expression of siRNA has emerged. The basic idea of this method is to clone the DNA double-stranded template sequence corresponding to the siRNA into the promoter of RNA polymerase III of the vector, so that the desired siRNA molecule can be expressed in vivo. The overall advantage of this approach is that it does not require direct manipulation of the RNA and achieves gene silencing for a longer period of time.2. Most of the siRNAs expressed by plasmid are initiated with Pol III promoter to start the sequence encoding shRNA (small hairpin RNA). The reason for choosing Pol III promoter is that this promoter always starts the transcription and synthesis of RNA at a fixed distance from the promoter, and terminates when it encounters 4-5 consecutive U, which is very precise. When this plasmid with Pol III promoter and shRNA template sequence is transfected into mammalian cells, this plasmid that can express siRNA can indeed down-regulate the expression of specific genes, and can repress both exogenous and endogenous genes. The advantage of using plasmids is that siRNA vectors are able to repress the expression of target genes for a longer period of time through the selective labeling of siRNA expression plasmids. And of course, since plasmids can be replicated and amplified, this significantly reduces the cost of preparing siRNA compared to other synthesis methods.3. Antibiotic-tagged siRNA expression vectors can be used for long-term inhibition studies. Through resistance-assisted screening, the plasmids can inhibit the expression of target genes in cells for several weeks or even longer. Meanwhile, RNAi-Ready expression vectors can also be integrated with retroviral and adenoviral expression systems (BD Knockout RNAi Systems), which can greatly improve the invasiveness of siRNA expression vectors to host cells, and completely overcome the obstacle of the low efficiency of transfection in some cells, which is an ideal tool for the realization of the transient expression of mammalian cells and stable expression of siRNAs.Synthesis template1. Synthesize two single-stranded DNA templates encoding shRNA, with RNA PolyIII polymerase transcriptional abort site at the back of the template strand, and BamH I and Hind III cleavage sites at both ends, which can be cloned into the siRNA vector polyclonal site between BamH I and Hind III cleavage sites.
2. 95 ℃, 5 min, slow annealing, DNA single-stranded to get shRNA DNA double-stranded template.V. Ligation and transformation1. Thaw 100 ul of receptor cells on ice.2. Take 5 ul of ligation product and add it to the receptor cells, gently rotate it several times to mix the contents, and leave it on ice for 30 minutes.3. Place the tube into a water bath pre-warmed to 42°C and heat-excite for 90 seconds. Quickly transfer the tube to an ice bath and allow the cells to cool for 1 to 2 minutes.4. Add 700 ul of LB medium to each tube and incubate for 1 hour at 37°C with shaking for recovery.5. Centrifuge at 4 000 rpm for 5 minutes at room temperature, and after discarding the supernatant, resuspend the cells with the remaining 100 μl medium and spread onto the surface of LB agar plate containing resistance. Note: The amount of cells should be adjusted according to the efficiency of the connection and the efficiency of the sensory cells.6. Place the plate at room temperature until liquid is absorbed.7. Invert the plate and incubate at 37°C. Colonies may appear after 12 to 16 hours.PCR identification and sequencing identificationDesign PCR primers on both sides of the inserted DNA double-stranded template coding for shRNA, and amplify fragments between 100-200 bp.

Caveat

1. Starting from the AUG start code of the transcript (mRNA), search for the "AA" sequence and write down the 19-base sequence at the 3' end of the transcript as a potential target site for siRNA. Some studies have shown that siRNAs with a GC content around 30%-50% are more effective than those with a high GC content.Tuschl et al. suggested not to target untranslated regions (UTRs) at the 5' and 3' ends when designing siRNAs because these areas are rich in regulatory protein-binding regions, and these UTR-binding proteins or translation initiation complexes may affect the binding of mRNAs by the siRNP endonuclease complex, thus influencing the effectiveness of siRNAs. The effect of siRNA.

2. compare the potential sequences with the corresponding genomic databases (human, or mouse, rat, etc.) and exclude those sequences that are homologous to other coding sequences/ESTs. For example, use BLAST (www.ncbi.nlm.nih.gov/BLAST/)3. Select a suitable target sequence for synthesis. Often multiple target sequences of siRNAs need to be designed for a gene to find the most efficient siRNA sequence.

4. A complete siRNA experiment should have a negative control. The siRNA used as the negative control should have the same composition as the selected siRNA sequence, but no significant homology to the mRNA. It is common practice to scramble the selected siRNA sequence, again checking the results to ensure that it has no homology to other genes.If the plan is to synthesize siRNA, then the 21 base sequence starting with AA can be provided directly and the manufacturer will synthesize a complementary pair of sequences. Note that usually synthesized siRNAs end in 3'dTdT, and if they are to end in UU they usually have to be specified. There are results showing that there is no difference in effect between UU ending and dTdT ending siRNAs. This is because this protruding end does not need to be complementary to the target sequence.

Common Problems

I. Successful points of siRNA operation

1. Design and test two to four siRNA sequences for each gene.

To find potential target sites, scan the AA sequences in the target gene. Record the 19 nucleotides at the 3’end of each AA as potential siRNA target sites. Potential target sites need to be analyzed by BLAST analysis of the GENBANK database to remove those target sites that are clearly homologous to other genes. If possible, siRNAs should be designed based on regions of mRNA low secondary structure.

2. Selecting siRNAs with low GC content

Ambion has found that siRNAs with GC content of 40-55% are more active than those above 55%.

3. purify in vitro transcribed siRNAs

Confirm the size and purity of siRNA before transfection. To obtain high purity siRNA, it is recommended to remove excess nucleotides, small oligonucleotides, proteins, and salt ions from the reaction by either glass fiber binding, elution (purification columns are included in the Ambion siRNA In Vitro Synthesis Kit to ensure siRNA purity), or by removing excess nucleotides, small oligonucleotides, proteins, and salt ions from the reaction by 15-20% acrylamide gel. Note: Chemically synthesized RNA usually requires running gel electrophoresis for purification (i.e., PAGE gel purification).

4. Avoid RNAase contamination

Trace amounts of RNAase will lead to failure of siRNA experiments. Since RNAase is prevalent in the experimental environment, such as skin, hair, all objects touched with bare hands or exposed to air; it is important to ensure that each step of the experiment is free from RNAase contamination.Ambion has a complete line of products in this area, such as RNAase removing sprays, swabs, and liquids, to detect and remove RNAase.

5. Healthy Cell Cultures and Strict Handling Ensure Reproducible Transfections

In general, healthy cells are more efficiently transfected. In addition, a lower number of passages ensures the stability of the cells used in each experiment. In order to optimize the experiment, it is recommended to use less than 50 generations of transfected cells, otherwise the cell transfection efficiency will decrease significantly over time.

6. Avoid applying antibiotics

Ambion recommends avoiding the use of antibiotics from the time the cells are planted until 72 hours post-transfection. Antibiotics accumulate toxins in the penetrating cells. Some cells and transfection reagents require serum-free conditions during siRNA transfection. In this case, comparative experiments can be done with both normal and serum-free media to obtain optimal transfection results. qiagen introduces reagents specifically designed for RNA transfection.

7. Selection of good transfection reagents for transfection of siRNAs

Selection of good transfection reagents and optimization of operation for the target cell type are crucial to the success of siRNA experiments. siRNA experiments require the selection of transfection reagents suitable for transfecting small RNAs (most of the current transfection reagents are designed for transfecting larger plasmid DNA rather than small RNA molecules, and the host ranges are different in size). Transfection Kit by Ambion, Transmessenger Transfection Reagent by QIAGEN are transfection reagents optimized for trans siRNA, which are ideal for you.

8. Optimize transfection and detection conditions with suitable positive controls

For most cells, the housekeeping gene is a better positive control. Transfect different concentrations of positive control siRNA into target cells (also suitable for experimental target siRNA), and count the reduced level of control protein or mRNA relative to untransfected cells 48 hours after transfection. Excessive siRNA will lead to cytotoxicity to the point of death. ambion offers a wide range of positive siRNAs for target genes.

9. Exclusion of non-specific effects by negative control siRNAs

A suitable negative control can be designed by disrupting the nucleotide sequence of the active siRNA. Care must be taken that it is compared for homology to ensure that there is no homology relative to the genome of the organism to be studied.

10. Optimizing experiments by labeling siRNAs

Fluorescently labeled siRNAs can be used to analyze siRNA stability and transfection efficiency. Labeled siRNAs can also be used for intracellular localization of siRNAs and double-labeling experiments (with labeled antibodies) to track cells introduced with siRNAs during transfection, combining transfection with down-regulation of target protein expression.

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