To follow up on your response, what sites are compatible with your lentivirus backbone? What promotor(s) do your LV backbones have? What is the approx time frame to generate this type of construct?
We have a series of lentiviral vector with different promoters like EF1a, CMV, UbC, PGK. Please open the following links for site compatibility or send us your vector map. The subcloning can be done within 2 weeks.
It appears that you produce the lentivirus after developing the lentivirus construct. Can you insert a gene of interest into a lentivirus backbone and then just provide me the DNA of the construct? I do not need it packaged?
Yes, the cost for a single subcloning is $550 if you have the plasmid DNA with compatible cloning sites. An extra fee will be charged if there are no compatible sites. We will just ship you the plasmid construct.
What would be the best vector for use in stem cells? Do you have a GFP marker? How long does it take to clone and package?
EF1a promoter would be the best choice for stem cell research. Our custom subcloning normally take 2 weeks and please let us know your exact vector design and we will be able to get it done in a timely manner.
What bacterial strains should I use to amplify the DNA for lentivirus packaging?
Methylation is a concern for viral packaging so we recommend E. Coli strain DH5alpha for DNA amplification. "Stabl3 Cells" from invitrogen can also be used for DNA amplification.
How should I store my lentivirus?
Aliquots should be made for the lentivirus and stored at -70 degrees Celsius.
How do I infect suspension cells?
For non-attached cells transduction, we normally spin down the target cells and resuspend the cells in viral supernatant in the smallest culture dish possible, such as a 24-well plate or 6-well plate, depending the number of the target cells. A few drops of fresh medium should be added if the cells are very sensitive to medium starvation. The cells then undergo incubatation overnight and fresh medium is added the next day for normal culture.
Transgene expression should be detectable after 3-4 days transduction.
How come the infected cells are not resistant to the antibiotic?
Cells usually need time to recover from the transduction step and the recovery time varies depending on the cell type. Cells are often stressed after transduction and they may be more sensitive to antibiotics, even if the cells have been successfully transduced with the resistent gene. This is why why we suggest the researchers DO NOT use antibiotic selection step on primary cells for cell immortalization experiments. Primary cells will die due to their senescence properties so selection is not required and these are usually very sensitive to antibiotics, regardless if they have resistant gene expression.
Antibiotic resistance is also dependent on cell density, and it can be thought as a level of dosage per cell. If the cells are highly dense, they are usually more tolerant than cells at lower density with the same antibiotic concentration. This may be relevant to the customers if they have done the following experimental design. The negative control and the experiment sample started out with the same cell density (ex. 50%). For the negative control, they did not add anything to the cells, while the experiment sample underwent transfection. Transfection is known to cause cell death and toxicity in certain cells and that may affect the cell density. Thus, after 48-72hrs, the cell density of the experimental sample will most likely be lower than the negative control because the negative control is growing normally. If the same antibiotic concentration is used on the two, the negative control may survive due to its higher density and the experimental sample will not survive, as the cell density is too low to thrive at the antibiotic concentration.
Here are two suggestions to resolve the dilemma.
1. Use a lower concentration of antibiotic in the first week after infection to allow the cells to recover. Afterwards, use the normal concentration of antibiotic for selection.
2. Do a transduction with GFP reporter on the cells of interest. If successfully transduced, the cells will be glowing green. These cells can then be grown and evaluated with a killing curve for antibiotic resistance.
How do I find the optimal concentration of antibiotic for the transfected cells? How do I do a killing curve?
A killing curve should be done for untransfected cells, but the curve for transfected cells is likely to be different, and it will depend on the strength of the viral promoter. The resistance is normally a little lower due to transfection stress. The promoter for antibiotic is often the SV40 early promoter, which is of medium strength. It is difficult to do a killing curve with transfected cells, but it is possible if the starting concentration on the transfected cells is 50ug/ml less than untransfected cells. Prepare 2x 6-well plates, one for the control, one for the experiment. In each of the 6 wells, have different antibiotic concentrations. Transfect the control and the experiment. The concentration that cause death in the negative control and show resistance in the experimental sample is the optimal one. It’s very important to monitor the cells daily and select the lowest amount that can kill the all the control cells within 4 days.
What types of tags are available in the lentivirus vector? Where can I find the map?
Please go to the link below to find our vector offerings and the maps:
If you want a specific tag that is not listed on our website, we can get that arranged through a custom service as well.
Do we receive the subcloned lentiviral vector in addition to the virus?
If the subcloning service was requested in addition to viral production, you will receive the newly formed vector along with the viral particles.
If a high-titer custom lentivirus was ordered, do we do the lentiviral titration after a freeze-thaw or straight after production before the freeze-down step?
We do the titration after aliquoting and freeze-down. Thus, the titer should be accurate for the customer after they thaw the finished product for the first time.
Which virus has your lentivirus expression system been derived from? Is it HIV?
Our lentivirus expression system is derived from Human HIV-1 Virus. It employs third generation self-inactivating recombinant lentiviral vectors with enhanced biosafety features and minimal relation to wild-type Human HIV-1 Virus.
Can I choose the particular LV backbones (e.g. LV013; promoterless vector) for the sub-cloning service?
Yes, our sub-cloning service applies to any of our vector backbones. Feel free to contact us at firstname.lastname@example.org for any assistance with your project design
How do you verify the titer?
We use our Cat#LV900 - qPCR Lentivirus Titration(Titer) Kit. This kit quantifies a proprietary region of the lentiviral 5-UTR.
How much DNA do I need to provide for the LV002- services? (DNA amplification required)
We request 3-5 ug of DNA to allow enough for amplification and restriction digestion to check that the amplified DNA band pattern matches the original plasmid pattern.