How to consistently produce high titer lentivirus?

Lentivirus production is not a complicated process. However, how to product high titer lentivirus relailably 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 (trans-gene plasmid and packaging plasmid mixture): 

• Trans-gene lentivectors: Promoter selection, expression cassette arrangement, enhancer elements, lentivector size all affect the produced lentivirus titer. 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. CAT#: HT-Pack.
• Plasmid quality: the propagated and purified all plasmids must in high quality in order to produce high titer lentivirus. 

2. Lentivirus production procedures: 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 (CAT#: TLV-C).

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 transfection 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. In order to select for long term expression, 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),  Hygromycin (Hygro), or Zeoin (Zeo). 

Depend your preference with the selection marker, you pick the product containing your desired antibiotic marker, Bsd, or Puro or Neo or Hygro or Zeo.

Each cell type has its optimal antibiotic selection concentration for each antibiotic type. Puromycin and Blasticidin has the “fast killing feature” (often in about one week). Most cell types are extremely sensitive to puromycin and the stable cell line can be generated in one week.

Depends on brand name, the antibiotic potency, also different culture conditions, the selection concentration for your cells has to be tested in the killing curve for each brand’s antibiotic. 

Gentarget provide cell culture ready antibiotic solution stock, sterilized by 0.22um filter. More important, our antibiotic solutions are tested for its working concentration in some cell types. see each products for details, CAT#: P-anti; B-anti; G-anti; Hygro-Anti and Z-anti ;

Use Lentivirus Transduction Enhancer:

Some cell types are not susceptible to lentivirus and has very low transduction efficiency, such as the primary T cell, macrophages and hematopoietic stem cells. Therefore, the transduction enhancer is much needed in such cases. 

Gentarget developed a novel transduction enhancer (Trans-Up^TM). It is the poly-cations and non-ionic amphiphilic molecules mixture using a proprietary process. This nontoxic chemical-based enhancer increases the transduction efficiency by neutralizing the particles’ and cell surface charges and modulating cell membrane permeability, which condenses lentivirus and bridge into to cells. Whether for difficult-to-transduce cell type of just want to ensure high transduction rate, the Trans-Up can increase transduction rate by 2-8 folds depend upon cell types and original lentivirus transduction efficiency. see details in Product page (CAT#: T-Up)

Obtain high-titer lentivirus using “Lentivirus Concentration Kit”:

High-titer lentivirus is desired for some hard-to-transduced cell types, and for many in vivo applications. Gentarget developed a virus concentration kit. Virus is concentrated using the non-cell toxic PEG (Poly-Ethylene Glycol) precipitation method, without the tedious and time-consuming ultra-centrifugation process. The concentrated virus can be used directly for in vitro and in vivo application. 

The process is easily scaled up to accommodate larger supernatant volumes, increases the lentivirus titer (IFU/ml) by 10 to 100 folds, with recovery 50% to 90%. See details in product page, CAT#: LV-CONC