We have a series of lentiviral vectors with different promoters like EF1a, CMV, UbC, PGK. Please open the following link for site compatibility or send us your vector map. The subcloning can be done within 2-3 weeks.

Yes, see our Subcloning Service I if you have the plasmid DNA with compatible cloning sites and Subcloning Service II if there are no compatible sites. We will just ship you the plasmid construct.

EF1a promoter would be the best choice for stem cell research. We can customize vectors to include GFP. Our custom subcloning normally takes 2-3 weeks. Please let us know your exact vector design and we will be happy to look into this for you.

Rearrangement is a concern for viral packaging so we recommend our ProClone™ Competent Cells (E. coli strain DH5alpha) for DNA amplification.

Aliquots should be made for the lentivirus and stored at -70 degrees Celsius. Or alternatively, inquire about special aliquotting services at the time of order placement.

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 incubation overnight and fresh medium is added the next day for normal culture. Transgene expression should be detectable after 3-4 days transduction.

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 resistance gene. This is 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 of whether they have resistance gene expression. Antibiotic resistance is also dependent on cell density, and it can be thought of 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 experimental sample started out with the same cell density (ex. 50%). For the negative control, they did not add anything to the cells, while the experimental sample underwent transfection. Transfection is known to cause cell death and toxicity in certain cells and that may affect the cell density. Thus, after 48-72 hrs, 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. 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. 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.

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.

Please go to the link below to find our vector offerings and the maps: http://www.abmgood.com/Lentiviral-Expression-Systems.html

We can provide custom tags. If the subcloning service was requested in addition to viral production, you will receive the newly formed vector along with the viral particles.

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.

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.

Yes, our sub-cloning service applies to any of our vector backbones. Feel free to contact us at technical@abmgood.com for any assistance with your project design

We use our LV900 series - qPCR Lentivirus Titration(Titer) Kit. This kit quantifies a proprietary region of the lentiviral 5’-LTR.

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.

The maximum insert size is 8kb.