Frequently Asked Questions:Customized Service 
1. There are several viral gene delivery systems, including adenovirus, Adeno-Associated Virus, retrovirus, and lentivirus. Which viral vector should I use?
2. What is meant by replication defective?
3. What is the required biosafety level for using recombinant viruses?
4. What are the recommended storage conditions of recombinant viruses?
5. What are the differences between plaque formation unit (PFU), Vector Genomes (VG), Transducing Units(TU) and infectious unit (IFU)? Which one of these better reflects the amount of active virus used?
6. What's the difference between the two genes by using 2A, IRES and fusion expression or other linker to link?
7. What should I do if I can’t find the antibody to detection the protein that I am studying?
8. Which promoter to choose for shRNA? U6 or H1?
9. What if the expression level of targeted genes in cells is too low to detect the interference efficiency?
10. What are the advantages of using shRNAs versus siRNAs?
11. What is the difference between using shRNA viruses and shRNA plasmids?
12. Does shRNA have the same effect as the corresponding siRNA?
13. What is the function of the scrambled control?
14. You guarantee that the interference has a 70% reduction efficiency, but why was the reduction rate so low when I did the experiment?
15. What are the advantages of CRISPR/ Cas9-mediated knockout over shRNA-mediated knockout?

---

1. There are several viral gene delivery systems, including adenovirus, Adeno-Associated Virus, retrovirus, and lentivirus. Which viral vector should I use?
Different virus lines are better for some applications than others. Choosing the best virus to meet your experimental goals can be difficult, but we are here to help. Below is a chart with a general overview of our viral vectors. If you have more detailed questions, please e-mail us at sales@brainvta.com or click the Send Request button directly and we would be happy to discuss your experiment. 

Vector	Infection	Advantages/ Disadvantages	Expression	Expression Level	Insert Size
Adenovirus	Dividing and non-dividing cells	Non-integrating Wide host cell range Immunogenic	Transient	High	7.5 kb
Adeno-Associated Virus (AAV)	Dividing and non-dividing cells	Non-integrating Non-immunogenic Non-pathogenic	Stable	Moderate	ssAAV-up to 4.5kb
Lentivirus* ( HIV)	Dividing and non-dividing cells	Random integration Minimal immune response	Stable	Moderate	6.5kb
Retrovirus* (MMuLV)	Dividing cells	Integrates Immunogenic	Stable	High	6.5kb
Baculovirus**	Insect cells	Species-specific	1 mg/1x109 cells	High	No upper size limit

*Replication defective.
** Replication defective in mammalian cells. Replication competent in insect cells.
2. What is meant by replication defective?
Replication defective means that critical portions of the viral genome have been removed such that the viral vector can no longer replicate. The removal of these genes makes room for insertion of exogenous gene.
3. What is the required biosafety level for using recombinant viruses?
The recombinant viruses we made are all replication deficient. According to references issued by the NIH Office of Biosafety, recombinant viruses have been classified in biosafety level II for agents considered of ordinary potential harm, and you need BL-2 level facility to work with it.
4. What are the recommended storage conditions of recombinant viruses?
The virus stock should be aliquoted upon receipt and stored at -80°C, especially after CsCl or chromatography purification. Repeated freeze-and-thaw should be avoided, since it will cause significant decrease of titer.
5. What are the differences between plaque formation unit (PFU), Vector Genomes (VG), Transducing Units(TU) and infectious unit (IFU)? Which one of these better reflects the amount of active virus used?
1) PFU (plaque formation unit) represents the number of infectious or live viruses. PFU/ml, it was used to calibrate the virus titer by measuring the number of plaques after virus infection. It reflects the amount of working viruses in the preparation.
2) IFU (infectious unit) is equivalent to PFU.
3) VG (Vector Genomes). vg/ml, which refers to the genome copies per milliliter of the virus venom. VG does not reflect the amount of active virus in the preparation.
4) TU/ml: Transducing Units/ml, which represents the number of bioactive virus particles per milliliter of the virus venom, that is, the number of viral genomes that can infect and enter target cells. This is a common unit of measure for lentiviruses and can reflects the amount of working viruses in the preparation.
6. What's the difference between the two genes by using 2A, IRES and fusion expression or other linker to link?
1) 2A: The two genes connected by 2A were expressed separately, and the expression levels of the two genes is consistent. But when 2A is expressed, there will be a residue, and if the size of the target gene is smaller than the size of the residue, it will affect the expression of the target gene.
2) IRES: The two genes connected by IRES were expressed separately, but the expression levels of the gene that after 2A is weaker.
3) Fusion expression is commonly used for the location of a target gene.
4) We can also add linkers/spacers according to customer requirements.
Therefore, the client needs to choose the appropriate gene connection mode according to the experimental purpose.
7. What should I do if I can’t find the antibody to detection the protein that I am studying?
If you can’t find the antibody to detection the protein that you are studying, you can choose Flag or His to express with your target gene by fusion. But you need to confirm to fuse it with C-terminal or N-terminal.
8. Which promoter to choose for shRNA? U6 or H1?
The U6/H1 promoter is commonly used to express shRNA. However, theoretically, the expression intensity of U6 is higher than H1, so U6 is more commonly used. H1 is generally used to construct stable cell lines.
9. What if the expression level of targeted genes in cells is too low to detect the interference efficiency?
We will make a vector with overexpression of foreign genes and then transfect it to cells to increase the expression of target genes.
10. What are the advantages of using shRNAs versus siRNAs?
siRNAs are chemically synthesized and their use is restricted to experiments studying the impact of transient suppression of gene expression. shRNAs are carried by a plasmid or viral-based vector. The vector may carry an antibiotic-resistance gene or a reporter gene, which permits the selection of a stably transfected cell.
11. What is the difference between using shRNA viruses and shRNA plasmids?
Using shRNA viruses can efficiently deliver the shRNA into any mammalian cell including that are difficult (or impossible) to transfect. Additionally, viral transduction is a much more efficient than transfection.
12. Does shRNA have the same effect as the corresponding siRNA?
siRNA is structurally different from shRNA. siRNA is chemically synthesized. It is usually used at a concentration of 5um, so that the number of molecules entering the cell at once is very large (more than 8 power), and it can bind to the target without cutting, so its probability of effectiveness is close to 90%. Interferes by shRNA is to clone the interfering gene to the DNA, which is then incorporated into the cell by either viral integration (lentivirus) or non-integration (adenovirus, adeno-associated virus) before binding to the target sequence through a series of processes. This process is more complex and therefore less efficient than siRNA. In addition, siRNA, which is effective, is sometimes not suitable for shRNA.
13. What is the function of the scrambled control?
The scrambled control clone is constructed by cloning a scrambled sequence (one that does not match any genomic sequences) into shRNA vectors. It serves as a negative control to eliminate the potential non-specific effect induced by expression of the plasmid.
14. You guarantee that the interference has a 70% reduction efficiency, but why was the reduction rate so low when I did the experiment?
1) If there is no resistance gene, the effectiveness of the virus infection needs to be confirmed. Because the efficiency of interference is largely determined by the efficiency of infection.
2) Check PCR primers and CT values.
3) The endogenous expression of some genes is extremely low, and the CT value is close to or more than 30. Such genes with low expression have little significance for down-regulation, which belongs to the problem of preliminary experimental design, so it is suggested to replace target cells.
15. What are the advantages of CRISPR/ Cas9-mediated knockout over shRNA-mediated knockout?
shRNA knocks out the target gene at the mRNA level, while Cas9 knocks out the gene at the genomic level, completely eliminating the target gene expression in the cell.