abm‘s lentiviruses can infect both dividing and non-dividing cells, and will integrate stably into the host cell genome, ensuring long-term expression of the construct even in cells that are difficult to infect. abm’s proprietary packaging mixes and efficient protocol based on years of experience permit rapid production of recombinant lentiviral particles with titres up to 1010 IU/ml. We have successfully completed thousands of orders.
“Although there are alternatives for custom lentivirus production, I have found abm to offer the best price and service for the production of high titer lentivirus preps. If I have any special needs or concerns, they are always dealt with by the Technical Support Team. I have always been pleased with the product and will continue to use this service for my research.”
John Teem, Florida Department of Agriculture & Consumer Services, Custom Lentivirus Production
Gene synthesis & Cloning
|GENE SYNTHESIS & SUBCLONING# SERVICE||UNIT||CAT. NO.|
|Gene Synthesis||Per bp||C098|
|Subcloning Service I||1.0 μg||C096|
|Subcloning Service II *||1.0 μg||C097|
|Site-directed Mutagenesis *||1.0 μg||C047|
|* Additional Charge for Extra 1kb||N/A||C100|
# For full details regarding our Gene Synthesis and Subcloning options, please visit here.
Lentivirus Packaging Services
|ENOUGH DNA PROVIDED||UNIT||TITER||CAT. NO.|
|Custom Recombinant Lentivirus Packaging||10 ml||106 IU/ml||LV001|
|High-Titer Custom Recombinant Lentivirus Packaging||1.0 ml||107 IU/ml||LV001‑a|
|High-Titer Custom Recombinant Lentivirus Packaging||550 μl||108 IU/ml||LV001‑b|
|High-Titer Custom Recombinant Lentivirus Packaging||5 x 20 μl||109 IU/ml||LV001‑c|
|High-Titer Custom Recombinant Lentivirus Packaging||5 x 20 μl||1010 IU/ml||LV001‑d|
|DNA AMPLIFICATION REQUIRED||UNIT||TITER||CAT. NO.|
|Custom Recombinant Lentivirus Packaging||10 ml||106 IU/ml||LV002|
|High-Titer Custom Recombinant Lentivirus Packaging||1.0 ml||107 IU/ml||LV002‑a|
|High-Titer Custom Recombinant Lentivirus Packaging||550 μl||108 IU/ml||LV002‑b|
|High-Titer Custom Recombinant Lentivirus Packaging||5 x 20 μl||109 IU/ml||LV002‑c|
|High-Titer Custom Recombinant Lentivirus Packaging||5 x 20 μl||1010 IU/ml||LV002‑d|
|Custom Lentivirus Titration – qPCR-based (Detectable Range of 104-1010 IU/ml)||1 Sample||C099|
Lentivirus Custom Aliquoting
|SERVICE NAME||UNIT||CAT. NO.|
|Lentivirus Custom Aliquoting||Up to 10 Vials||C507|
*Minimum volume per vial = 20 μl
***For LV001, LV001-a, LV001-b, LV001-c and LV001-d, please refer to the Service Minimum Requirements for details about DNA submission requirements.***
- *For customer supplied vectors, abm is only able to guarantee successful virus packaging as verified by our qPCR-based lentivirus titration assay. We recommend that gene expression and any other desired vector functionality is verified by transfection of the cells in which the virus is to be utilized, prior to submitting your plasmids for packaging.
- **For customer supplied vectors, the customer must indicate prior to the service if any reporter expression will be expected or not. The expected reporter expression, e.g. GFP, should not require induction of expression. Due to differences in excitation/emission wavelengths for different fluorophores, we may not be able to provide infection images for fluorescent reporter expression other than standard GFP/RFP/mCherry.
- abm’s lentiviral vectors are 3rd generation lentivectors, and can be packaged into replication-incompetent lentiviruses with either 2nd Generation or 3rd Generation packaging systems.
- What sites are compatible with your lentivector backbone? What promoter(s) do your LV backbones have? What is the approx time frame to generate this type of construct?
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.
- It appears that you produce the lentivirus after developing the lentivector construct. Can you insert a gene of interest into a lentivector backbone and then just provide me the DNA of the construct? I do not need it packaged.
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.
- 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. 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.
- What bacterial strains should I use to amplify the DNA for lentivirus packaging?
Rearrangement is a concern for viral packaging so we recommend our ProClone™ Competent Cells (E. coli strain DH5alpha) for DNA amplification.
- How should I store my lentivirus?
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.
- How do I infect cells in suspension?
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.
- 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 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.
- 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: http://www.abmgood.com/Lentiviral-Expression-Systems.html
- If I want a specific tag that is not listed on your website, can I get that as a custom service? Do we receive the subcloned lentiviral vector in addition to the virus?
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.
- If a high-titer custom lentivirus was ordered, do you 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 LV900 series - qPCR Lentivirus Titration(Titer) Kit. This kit quantifies a proprietary region of the lentiviral 5’-LTR.
- 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.
- What is the packaging capacity for Lentivirus?
The maximum insert size is 8kb.
Khanna, S et al. “Oxygen-Inducible Glutamate Oxaloacetate Transaminase as Protective Switch Transforming Neurotoxic Glutamate to Metabolic Fuel During Acute Ischemic Stroke.” Antioxid Redox Signal. 10:1777-1785 (2015). DOI: 10.1089/ars.2011.3930 PubMed: 21361730 Application: Gene Delivery, Lentivirus
Somanna, NK et al. “Intratracheal administration of cyclooxygenase-1-transduced adipose tissue-derived stem cells ameliorates monocrotaline-induced pulmonary hypertension in rats.” Am J Physiol Heart Circ Physiol 307(8):H1187 (2014). DOI: 10.1152/ajpheart.00589.2013 PubMed: 25320332 Application: Gene Delivery, Lentivirus
Roy, A et al. “Regulation of cyclic AMP response element binding and hippocampal plasticity-related genes by peroxisome proliferator-activated receptor α.” Cell Rep 4:724–737 (2013). DOI: 10.1016/j.celrep.2013.07.028 PubMed: 23972989 Application: Gene Expression
Song, MK et al. “Polycyclic aromatic hydrocarbon (PAH)-mediated upregulation of hepatic microRNA-181 family promotes cancer cell migration by targeting MAPK phosphatase-5, regulating the activation of p38 MAPK.” Toxicol. Appl. Pharmacol. 273:130-9 (2013). DOI: 10.1016/j.taap.2013.08.016 PubMed: 23993976 Application: Gene Expression