How to consistently produce high titer lentivirus?
Lentivirus production is not a complicated process. However, to consistently produce high titer lentivirus is not a simple task. It involves many steps. And each step could comprise the virus’s quality. Two critical consideration for producing high titer lentivirus are:
1) Lentivector system: include trans-gene (expression) plasmid and packaging plasmid mixture:
Trans-gene lentivectors: Gentarget develops all its expression lentivectors in house with the optimized components to maximize virus titer and its packaging efficiency.
Packaging plasmid mixture: Gentarget uses three packaging plasmids for high bio-safety consideration. Three plasmids are carefully mixed in best ratio to maximize the packaging efficiency. The packaging plasmid / trans-gene lentivector mass-ratio is mixed in best ratio during the transfection step.
Plasmid quality: the propagated and purified all plasmids must in high quality in order to produce high titer lentivirus.
2) Lentivirus production procedures: include production cell line, plasmid transfection, virus production protocols:
Virus producing cell line:
Cell type: most common used cell line is HEK293-T cells as most trans-gene lentivector adapted the SV40-Ori element. Depends on your trans-gene lentivector structure, the virus production cell lines can be a cell line stable expressing the episomal enhancer for your trasns-gene lentivector. For example, a ENBA1 stable cell line will produce high titer lentivirus for lentivector containing OriP element. A few other genes that stably expressed in production cell line (like, XPO1, TNIP1) will also increase the packaging efficiency. Gentarget’s lentivirus production cell line are tested for high titer virus production.
Cell passage: High titer virus only can be produced in small cell passage number, and cells must cultured in “happy” status.
Transfection: The trans-gene lentivector and the packaging plasmids must be efficiently and evenly delivered into virus producing cell line for high titer virus production. The most common used method is lipid based transfection. The transfection rate is highly depends on lipid’s delivery efficiency. Gentarget produce a high efficient, lipid based transfection reagent (CAT#: LP4K) for the highest transfection rate. (see product below, or click to see the Product Manual). This lipid does not need DNA/lipd mixture optimization, best for large plasmid transfection, like lentivectors. It produces thousands of high titer lentivirus with high reproducibility. This tranfection reagent is also good for any plasmid transfection (not necessary for lentivirus production) in most cell types and best the for large plasmid delivery. Gentarget provide this lipid product for all customers.
Optimized lentivirus production procedure: Gentarget optimized its lentivirus production SOP. Each process step is controlled best for the highest virus titer production, the medium change time, the virus harvest time, the processing temperature, the post-production process, and so on.
How to select antibiotic marker in lentivirus products?
One important feature of lentivirus is its long-term expression / knockdown because it integrates into cell’s genome. To select the positive transduced cells, lentivirus often contains a fluorescent marker (for cell sorting) or /and an antibiotic marker (for killing selection).
GenTarget’s products are made with different antibiotic selection marker, Blasticidin (Bsd), Puromycin (Puro), Neomycin (Neo), or Hygromycin (Hygro).
Depend your preference with the selection marker, you pick the product containing your desired antibiotic marker, Bsd, or Puro or Neo or Hygro.
Each antibiotic has different killing curve in each cell type, which have to be tested prior to the antibiotic selection. Puromycin and Blasticidin has the “fast killing feature” (often in about one week). Most cell types are extremely sensitive to puromycin. So, you select the product with the antibiotic marker bets for your cell selection.
Depends on brand name, the antibiotic solution or powder has different potent. And different culture can affect the cell’s sensitivity to antibiotic. So the killing curve need to be tested for each brand’s antibiotic for each cultured cell type. The following table showed the best antibiotics concentration to use in some cell types.