Guide to Virus Selection & FAQ
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Guide to Virus Selection & FAQ
Guide to Virus Selection & FAQ
Guide for Selection of Gene Regulatory Tools
FAQ：
Q1、How to choose between lentivirus vectors and adenovirus vectors?
Answer：Both lentivirus vectors and adenovirus vectors are excellent gene regulation tools, each with its own advantages. The selection is mainly based on different experimental purposes, which can be judged by referring to the following aspects：
（1） Insert the size of the destination sequence：The packaging capacity of the lentivirus vector is within 2kb to ensure titer，Adenovirus packaging capacity within 5kb can ensure titer；If the size of the target sequence exceeds the carrier capacity, the titer will decrease as the size of the target sequence increases；
（2）Is it necessary to construct a stable cell line：Slow virus vectors can randomly integrate foreign genes into the genome of target cells and stabilize inheritance，Can be used to construct stable cell lines，Of course, instant transfection is also possible；Adenovirus, on the other hand, does not integrate its genome and is only suitable for transient transfection；
（3） Target cell type：Cells such as neural cells and some primary cells that are difficult to infect and do not require the establishment of stable transfection strains in the future are recommended to use adenoviruses, which have a much higher infection efficiency than lentiviruses.
Q2：How to save after receiving the virus?
Answer：If the virus is used for a short period of time after receiving the virus solution, it can be stored at 4 ℃ and preferably used up within a week；If long-term storage is required, it can be packaged and placed at -80 ℃ as needed to avoid repeated freeze-thaw during use；If stored in a liquid nitrogen tank, a dedicated storage tube needs to be replaced。In addition, if the virus has been stored for more than 6 months, Hanheng Biotech recommends retesting the virus titer before use。
Q3：How to dilute the virus during use?
Answer：When diluting a virus for use in a laboratory setting, it's important to follow proper safety protocols and handling procedures. Here are general steps for diluting a virus:
Thawing: If the virus is stored frozen, remove it from the freezer and thaw it slowly at room temperature or in a refrigerator. Avoid rapid thawing, as it can damage the virus particles.
Diluent: Prepare an appropriate diluent for the virus. This could be a buffer solution such as phosphate-buffered saline (PBS) or a suitable cell culture medium. Ensure that the diluent is compatible with the virus and the target cells.
Taking lentivirus as an example：To achieve a 1x10^7 TU/mL titer from a 1x10^8 TU/mL original titer, you would need to mix 10 μL of the original virus with 90 μL of diluent, resulting in a total volume of 100 μL.
Q4：What is MOI?
Answer：MOI，multiplicity of infection，it refers to the virus's ability to infect cells，the higher the MOI value required by the cells, the more difficult the cells are to be infected.The ratio of the number of virus particles to the number of cells required for 80 % of a cell to be infected is usually used as the MOI value of the cell.
The MOI is calculated by dividing the number of infectious agents (e.g., viral particles) by the number of target cells. For example, an MOI of 1 indicates that there is, on average, 1 infectious agent for every target cell in the culture.
Related calculation formula：
The amount of virus added per hole（μL）=MOI ×cells / virus titer（TU/mL or PFU/mL）×1000
MOI=（virus titer × Virus volume）/ cells
Q5：What is the amount of cell inoculation for viral infection ?
Answer：The amount of cell inoculation was adjusted according to the cell size and the speed of proliferation, so as to ensure that the cells just grew to the bottom of the culture dish 2-3 days after infection.For most cell lines : the passage cycle is 2-3 days, the density of the cell plate is maintained at about 30-50 % during infection, and the cell confluence is about 90 % after 48 h of cell proliferation.；For some primary cells : due to the slow growth of cells, can increase the degree of convergence to about 50-60 % at the time of inoculation, but to ensure that the cell confluence reached 90-100 % after 2 days of infection.；For non-dividing cells : such as neuronal cells, they no longer proliferate after inoculation, and can be inoculated at a 100 % confluence.
Q6：How to determine the best time to add the virus to the cell ?
Answer：It takes 48-72 h to observe the gene expression carried by the virus after lentivirus infection, and 36-48 h to observe the gene expression carried by the virus after adenovirus infection. The virus should be added when the cell confluence is 30-50 % and the cell is in good condition.
Q7：After the control virus or the target virus infects the cells, the cell morphology changes or cell death ?
Answer：First, determine if the cell or virus is contaminated.；Secondly, determine whether the MOI value of virus infection is too high, adjust the MOI value, and observe the cells at 4 h, 8 h and 12 h after infection. If the cell state is found to be deteriorated, the virus infection culture medium needs to be replaced with fresh complete culture medium.；After excluding the above factors, the cell state is still not good, try to increase the serum content, and observe whether the cell state is improved.
Q8：How to improve the efficiency of virus infection to cells ?
Answer：The efficiency of virus infection on cells is affected by many factors, such as virus activity, cell state, MOI value, and infection time.
①Virus activity : Thawing virus must be carried out on ice, try to avoid repeated freezing and thawing. Stored at-80 °C for more than half a year, it is necessary to retest the titer.
②Objective cells : First, a pre-experimental test was performed to see whether the viral vector was suitable for infecting the target cells. For suspended cells, the method of flat-angle centrifugal infection can be used to reduce the volume of virus infection, thereby improving the efficiency of infection.
③MOI value : MOI gradient groping experiment was carried out to find the optimal MOI value.
④Infection time : too early fluid replacement will lead to decreased infection efficiency ; if the medium is changed too late, the damage to the cells is greater. If the cell state is better, the medium change time can be appropriately prolonged to further improve the infection efficiency.
⑤Auxiliary agent : A certain concentration of polybrene can be added according to the situation. Different cells have different sensitivity to polybrene. The appropriate concentration can be screened in the range of 1-10 μg / mL, and the cells have no obvious toxic reaction within 24 h.
Q9：Which experiments can choose AAV virus ?
Answer：AAV virus is an efficient gene delivery tool in vivo, which is suitable for animal experiments in vivo. However, because of its capacity and expression characteristics, AAV is not suitable for experiments with large genes or rapid expression after infection, and the rest are generally applicable.
Q10：How should AAV virus be diluted and preserved ?
Answer：If the virus is used in a short time after receiving the virus solution, the virus can be stored at 4 °C and preferably used within a week ; if it needs to be preserved for a long time, it can be placed at-80 °C as needed to avoid repeated freezing and thawing during use. If it is to be stored in a liquid nitrogen tank, a special storage tube needs to be replaced. If dilution is required during use, PBS or normal saline is used for dilution on demand.
Q11：AAV injection in mice, how much virus is enough ?
Answer：There are many factors affecting the total amount of AAV injection, the more important are serum type, target tissue type, injection method（Local or whole body）、animal model（Like a rat or a mouse）etc，for the injection method reported in the literature, it is best to set several different gradients.For example, AAV9 infected mouse heart tissue（cardiac muscle cell），Tail vein injection or myocardial multi-point injection can be used.Tail vein injection recommended1011 vg/mL，100μL；Origin injection is recommended1012 vg/mL，20 μL。
Q12：What methods can AAV be injected into animals for infection?
Answer：According to the specific needs of experimental infection, it can be divided into systemic administration and local administration.Systemic administration, such as tail vein injection, delivers virus particles to the whole body through blood circulation, but this method has a good effect on liver infection, while it has a poor effect on other tissues and organs, especially on brain tissue. Because of the blood-brain barrier, it is necessary to cooperate with specific serotypes to achieve brain tissue infection through tail vein injection.；Second, local administration, such as brain stereotactic injection, intramuscular injection, etc. This injection method can infect local tissues more efficiently, has good specificity, and the amount of virus used is small. However, for the injection of some tissues and organs, surgery or special equipment is required, and the operation is difficult.
Q13：How long can we observe the effect of virus infection after AAV injection ?
Answer：AAV is a single-stranded DNA virus, which needs to form a double-stranded appendage to infect cells for gene expression.At 1-2 weeks, the target sequence is also expressed, but at this time, it is in the stage of gradual accumulation of expression, and the expression level is low. Therefore, it is generally recommended to detect the expression of the target gene after 3-4 weeks.
Q14：How to detect the overexpression of target gene after AAV injection ?
Answer：After 3-4 weeks of AAV virus injection, the detection began. Overexpression can be detected at the protein level by immunohistochemistry, immunofluorescence and wb, but usually the antibody used is relatively high. In order to facilitate the detection, a flag dial can be fused on the protein when constructing the overexpression vector, and the detection can be directly detected by commercial flag antibody detection, or the change of transcription level can be directly detected by qpcr.；For the detection of gene interference, the down-regulation of transcription level is generally only detected by qpcr.
Q15：Can tissue-specific infection be achieved with AAV ?
Answer：Yes.Tissue-specific infection can be achieved by AAV site-specific injection and specific serotypes. Combined with specific promoters, it can be fined to the specific expression regulation of specific cells.
Q16：What is VP、PFU、IU?
Answer：Different concepts are due to different titer determination methods. These methods include two categories.：physical method（VP）And biological methods（GTU、PFU、TCID50）。
VP：viral particl，is“live”（Infectious） and “fall”（Non-infectious）total amount of adenovirus particles.
PFU：plaque formation unit，It is infectious“live”total amount of adenovirus particles，That is, the adenovirus gets the active unit.
IU：infectious unit，Same as PFU，is another adenovirus active unit.The determination method is TCID50 method.
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