1. Which promoters can drive WZ Biosciences’s vector-based shRNAs?

In WZ Biosciences, multiple promoters can be used to drive shRNA expression in cells, including Pol III promoters, U6 and H1, and Pol II promoters, ubiquitous promoters (such as CMV, CAG and EF1a, etc.) and tissue-specific promoters (such as hsyn, TBG and cTnT, etc.).

2. Can human U6 and H1 work in rat, mouse or other mammalian cells?

In general, human H6 and H1 should all work in human, mouse, rat and other mammalian cells.

3. Do you guarantee knockdown efficiency?

For the set of shRNA constructs, we guarantee that at least one shRNA construct will generate ≥70% knockdown of every target gene(determined by qPCR), under the condition of transfection efficiency of >80% in target cells. If none of them can reach this knockdown efficiency, we will reconstruct new shRNA plasmids for free, which requires an additional turnaround time. If none of the re-constructed shRNA vectors still can’t achieve this knockdown efficiency, it may be associated with the target gene, we only charge you one time.

4. For every target gene, what you suggest for shRNA cloning services?

For every target gene, we suggest that you can send us you validated shRNA sequence or choose our the set of shRNA constructs.

5. How can I monitor transfection efficiency?

Our shRNA vectors contains GFP or RFP reporters which allow stimatanous transfection monitoring.

6. Do you construct shRNA vectors with other shRNA destination vectors purchased from other company?

Yes, we do. You just need to send us your desired shRNA vectors.

7. How long is the turnaround time for Vector-based shRNA?

Vector-based shRNA often need take 2-3 weeks.

8.What are the advantages of viral vectors used for gene delivery?

Viral-based shRNAs are ideal for hard-to-difficult cells, such as primary cells, fibroblast cells and dendritic cells (DC).

9. What materials and information do I need to provide for site-directed mutagenesis?

The materials to be provided by you:

If you will send your own vector for mutating or cloning, please send us at least 5 ug DNA or glycerol stock;

The information to be provided by you:

①Gene Name, sequence(pre-mutation sequence) and sequencing results;
②Description of sites/base pairs to mutate;
③Name of desired viral vector backbone or name of customer-provided vector.

10. How do you define one mutation?

One mutation is defined as any base changes within 6bp.

11. If I need to change the destination vector while mutating, how do you charge?

If you need to change the destination vector while mutating, additional cloning cost will be required.

12. If the customer don't know restriction sites at both ends of the insert sequence and plasmid map, but can provide the sequence and sequencing primer, can we undertake this project?

A: Yes, but we need to sequence to verify the sequence near the restriction site before cloning.

13. Can we provide glycerol bacteria as subcloning template?

A: Not recommended, because glycerol bacteria is not as stable as plasmid, but if glycerol bacteria is the only option, we can also try to do it.

14. The template was previously constructed by WZ Biosciences. Do you still have this plasmid, or do you need us to send it again?

A: We usually keep the constructed plasmid for 1 year. If the previous order has been completed for more than 1 year, but not more than 5 years, we may still have this plasmid, but we need to check with our production team.

15. We have primers, do we need to provide them or do you design and synthesize them yourself?

A: We will design and synthesize primers for cloning by ourselves. You only need to send us cloning template and let us know your desired gene sequence.

16. I have a complicated cloning scheme. Do I still need to design?

A: You just need to provide us template sequence, template plasmid and final sequence, our production team will design the best cloning scheme for you.

1. What is adeno-associated virus (AAV) & recombinant AAV(rAAV) ?

The adeno-associated virus (AAV) is a small, icosahedral and non-enveloped virus that belongs to Parvoviridae family. Helper virus such as Adenovirus or Herpes virus is usually required for a productive infection to occur. AAV does not encode its own polymerase so its replication process relies on host cell polymerase activities. Recombinant AAV is the artificial AAV without any AAV rep and cap genes which encode viral replication and structural proteins, respectively. In rAAV, rep and cap are replaced with a gene or construct of interest flanked by the ITRs for replication and packaging. Efficient packaging of rAAV can be performed with constructs ranging from 4.1 kb to 4.9 kb in size.

2. What’s the Biosafety requirement for using AAV?

Recombinant AAV constructs produced in the absence of a helper virus and encodes no tumorigenic gene can be handled in Biosafety Level 1(BSL-1) facility. Otherwise, it should be handled as biohazardous material under Biosafety Level 2 (BSL-2) containment.

3. Is the recombinant AAV safe?

To date, AAV is not linked to any human disease. For wild type AAV, replication is at extremely low efficiency, without the presence of helper virus, such as adenovirus. The recombinant AAV (rAAV) composed by several plasmids (cis plasmid, Helper plasmid, rep/Cap plasmid). Cis plasmid and Helper do not share any regions of homology with the rep/cap-gene containing plasmid, the likelihood for a recombinant AAV to replicate is theoretically impossible.

4. What’s the cloning capacity for recombinant AAVs?

AAV has a packaging capacity of ~4.7Kb. When the length of inserted DNA between the 2 ITRs is close the maximal allowed, i.e., 4-4.4Kb, the packaging efficiency decreases significantly. For instance, for gene over-expression from cDNA, since the CMV-poly(A) element is about 1Kb, so the maximal allowable cDNA length is about 3K, whereas if GFP co-expression ( about 2Kb) is considered, the allowable capacity is about 1-1.2 Kb.

5. What AAV serotype do you provide?

We currently provide AAV serotype AAV1，AAV2，AAV5，AAV6，AAV7，AAV8 &，AAV9，AAV rh10，AAV retro，AAV ANC80，AAV DJ & AAV DJ-8，AAV PhpB & AAV PhpeB，AAV 7m8 and AAV shh10.

6. What are the advantages of gene delivery by rAAV?

rAAV has the capacity to produce high titer virus in dividing and non-dividing cells and potential for long-term gene transfer with minimum immnunogenicity.

7. How stable are AAV vectors? How should they be stored?

Purified AAV vectors are highly stable at temperatures of 4 C or less. We recommend aliquoting upon receipt and storing stock at -80C for long term storage.

8. What does a customer need to provide for the custom AAV service? & Expected yield?

Please provide us with plasmid DNA for the specified gene, vector map and its sequence information. For gene silencing service, we need the exact shRNA sequence to be constructed into recombinant AAV vector if no plasmid available. Normally, expected yield is between 1x10^12-13 vp/ml, with about 500 ul viral stock will be given. However, special quantity/titer can be made upon request.

1. Hello, the cells I infected are trophoblast cells. 100,000 cells were inoculated in a six-well plate firstly, then virus (MOI=40) was added after 16 hours of attachment. 30 hours later, the cells became full. I would like to ask: After virus infection, the cell production of the experimental group is similar to that of the control group. Is it normal?

If the state of cells infected with adenovirus is not significantly different from that of the uninfected group, it means that the virus has no obvious toxic effect on cells. Therefore, it may not be that the number of viruses is insufficient. Non-adenovirus packaging cells will not have any obvious characteristics after being infected by adenovirus. If you add the virus to the culture dish after diluting the virus with the medium in advance when adding the virus, then the high MOI value (causing the cells to float) is relatively high for the target cells. Therefore, don’t worry, you can first check whether there is protein expression.

2. Is it necessary to replace the fresh culture medium after adenovirus infects cells?

Whether it is necessary to replace the fresh culture medium depends on the amount of virus added. When the amount of virus is large, it will be toxic to the cells: if there is a change in the cell state during the microscopic examination, the fresh medium needs to be replaced in about 4-8 hours; If there is no obvious change in the cell state, there is no need to replace the fresh culture medium after the virus infects the cell. Or, if you are worried that the virus will be toxic after acting on the cells for a long time, you can also replace the fresh culture medium after 12 hours.

3. Why the cells are all floating after 4-6 hours of adenovirus infection?

Three main reasons can explain the phenomenon above:

1) Cell status: The cells plated before infection must be healthy cells;

Improper operation: When adding virus, if the local virus concentration is too high, it will cause cell death. Therefore, we recommend mixing the virus and medium before adding.

If the 2 points above are both correct, then the amount of virus added is too large.

4. What is the basis for the amount of adenovirus virus when infection the cells?

Selecting the optimal virus amount is the key for getting the best experimental results.
Insufficient amount can’t reach 100% infection efficiency; too high amount will cause toxicity to cells. So how to determine it? Multiplicity of infection (MOI) plays a decisive role. MOI is the ratio of virus to infection cell number. The number of adenovirus receptors on cell surface is different in different cell lines, which determines that the MOI of different cell lines will be different. Generally, for susceptible cell lines, the MOI range used is 10-100. However, for some hard-to-infect cell lines, MOI may need to be as high as 1,000. For most cell lines, the optimal MOI range is very narrow. We recommend you to read the relative literature, or to test the best MOI by infecting the target cells with the adenovirus of the reporter gene before the formal experiment.

Calculation formula for optimal virus dosage
The amount of virus pfu=optimal MOI *cell number/ virus titer
For example, if the optimal MOI of target cell is MOI=10, 106 cells will be infected, then 107pfu virus are needed. If the virus titer is 1×1010 pfu/mL, then 1ul will be used for the experiment.

5. How long can the expression of adenovirus be detected at the gene level and protein level after infecting cells? Is there a difference in the detection time between secreted proteins and non-secreted protein?

The target gene carried by adenovirus expresses quickly. If you want to detect gene expression at the gene level, then perform the test between 12-24 hours of virus-infected cells; if you want to detect the gene expression at protein level, then perform the test between 48-72 hours of virus-infected cells.

For secreted protein and non-secreted protein, the detection time is the same. If you worried about protein secretion, you can do western blot together with the medium.

6. The adenovirus seed is contaminated by bacteria, there is no extra virus seed, and there is no constructed vector, what should I do?

The diameter of adenovirus is 90-100nm, and the diameter of bacteria is much larger, so it can be filtered with a 22um filter. If your virus load is very low and you are worried that you will lose too much after filtering and cannot proceed with the subsequent amplification work, you can directly amplify the contaminated adenovirus firstly. Of course, adenovirus contaminated with bacteria are obtained after amplification. But the amount of virus is increased, then use a 22um filter for filtration.

7. How to identify the target gene that adenovirus delivered?

To identify as follow:

①Design primers for genes, which can also be used as amplification and sequencing;
②Virus is the template which is treated with proteinase K. If it is an unpurified virus, it needs to pass through a purification column to remove the medium components.
③Perform PCR with the processed virus as a template;
④Sequencing the PCR products with PCR primers.

1. When to choose lentivirus？

Since lentivirus has a broad host-range, it can infect both dividing cells and non-dividing cells. Lentiviruses can be used as viral vectors in in vivo and in vitro studies. In addition, lentivirus is also an ideal virus for constructing stable transgenic strains.

2. What is the size limit for the ORF that is to be cloned into the lentivirus vector?

In general, lentiviral vectors can accommodate 8 kb inserts. However, when the inserted ORF gene is larger than 4 kb, the packaging efficiency of the virus will be greatly reduced, which will affect the virus titer and even gene expression.

3. What is the amount of cell inoculation used for lentivirus infection?

Inoculate target cells in good condition into a 24-well plate. The cell concentration is generally 1×10⁵/mL cells. Many factors are need to consider when calculate the number of cells to be inoculated, including the cell viability, status, growth speed, division factors, etc., generally to ensure virus infection It is better for the cell confluence rate to reach 50%-70%, when to ensure virus infection.

4. What is MOI?

MOI (multiplicity of infection), the multiplicity of infection, is the ratio of the number of virus particles to the number of cells at the time of infection. It is generally believed that MOI is a ratio without a unit.

5. Can lentivirus express genes stably?

Yes. Lentiviruses can integrate foreign genes into the host genome and will not be lost during cell division and passage. Therefore, the foreign genes can be stable integration and long-term expression.

6. How long is the lentivirus packaging cycle in WZ?

If custom can provide the constructed lentivirus vector, the packaging cycle is around 2 weeks, 5 weeks are need including preliminary vector construction and packaging.

7. After the lentivirus infects the cell, when does the peak expression of the target gene can be observed?

After infecting the target cells with lentivirus, the expression of the target gene can reach to the peak at 72-96h generally, but for some special cells, such as cells with slower proliferation and passage, it takes longer f to reach the peak expression.

8. How to improve the lentivirus infection efficiency?

Good cell growth and a suitable MOI value during infection are the guarantee for high infection efficiency. Generally, the infection efficiency of the virus can be improved by increasing the MOI value during infection. If necessary, the adjuvant agent ADV-HR can be added during infection to improve the infection efficiency.

9. After lentivirus infection, why the cell condition is very poor, and even death?

Lentivirus can cause certain toxicity to cells, and different cells have different tolerance to toxicity. It is recommended to reduce the MOI, to increase the confluence rate of plated cells (up to 70%) during cell preparation and adjust the amount of virus added during infection. In addition, you can also change the medium 4 hours after the infection, and continue the culture and observation with fresh complete culture medium.