Improve lentiviral production using Lipofectamine 3000 reagent

LentiVirus is extremely versatile and efficient transfection method to work in more biologically relevant cells.  However, they can be difficult to produce. Invitrogen™ Lipofectamine™ 3000 Transfection Reagent can help improve lentivirus production.

In addition to being a top performing reagent for difficult cell types, Invitrogen™ Lipofectamine™ 3000 Transfection Reagent is a highly efficient, cost-effective tool for lentiviral production. This versatile reagent enables high viral titers even with genes that are large or difficult to package.

During this Ask the Experts session, we will be discussion the challenges associated with lentivirus production, how Lipofecatmine 3000 can help as well as insider tips on how to improve lentivirus production.

Please take this opportunity to ask our expert your questions about improving lentivirus production.

Questions may include topics like:

  • Achieving higher titers
  • Working with larger gene sizes
  • Saving time and using a simpler protocol

This Ask the Expert session is sponsored by Thermo Fisher Scientific and hosted by Xin Yu. Xin is an R&D scientist with more than 10 years of experience working on in vitro transfection . She was one of members who developed Lipofectamine® RNAiMax, LTX and 3000 reagents. She was the key person in conducting the App Note – LentiVirus Production by using Lipofectamine® 3000.

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Question 1

I need to package a large gene, which is around a 4-5Kb lentivirus. I used PEI as the transfection reagent, but it gave me very low or almost no lentiviral particles. I was interesting in trying Lipofectamine 3000, do you have any suggestions?

Lipofectamine 3000 has the capability to package not only the standard 1-2Kb sized genes, but can also package large genes greater than 4-5 Kb, such as CRISPR Cas9. This reagent outperforms other reagents, including PEI for packing large genes. Here are some suggestions for using the invitrogen ViraPower™ packaging system: Example (6-well plate format): If … Continued

Question 2

What is the seeding density that is recommended for using Lipofectamine 3000 to produce lentivirus?

Unlike other commonly used transfection reagents that are used in Lentivirus production, Lipofectamine 3000 actually requires a high cell density (cell type: 293T or FT) at the time of transfection. The recommended density should be 95-99% at the time of transfection. Total: 0 0 0 0

Question 3

Is it comparable to package lentiviral particles using the forward and reverse transfection methods?

Yes, the two methods are comparable. Both of the transfection procedures are the same, however, the number of cells for seeding is different. Cell seeding density for reverse transfection method is 3 times higher than forward transfection. For example in a 6-well plate, 1.2×10^6 cells per-well are needed for forward transfection versus 3.6×10^6 cells for … Continued

Question 4

Is it ok to use antibiotics in the transfection media when using Lentivirus?

We always suggest our customers to replace cell media by adding fresh media without antibiotic (pen/strep) at the time of transfecting cells. We also have done some studies with and without antibiotic-containing cell media during regular DNA transfection procedures. We have found that antibiotics (pep/strep) do not affect the ability of lipofectamine reagents to transfect … Continued

Question 5

What are some advantages/disadvantages to using Lentiviral vs. other transfection methods?

For hard to transfect, non-dividing cells, Lentiviral transfection offers a high efficiency solution for attaining good expression levels. However, the time and difficulty associated with lentiviral production makes this method difficult and time consuming. In addition, a proper safety-level lab is necessary for production and use of the virus. An important note is that lentiviral … Continued

Question 6

Do I need to change media 6-16 hours post-transfection during the lentivirus production process?

No, it is not necessary if you are going to concentrate the lentiviral particles 24-48hrs post-transfection after harvesting cell supernatant twice. However, if you are going to use crude lentivirus in your next step and transduce target cells, the remaining DNA/reagent complex might be toxic to your cells. If you don’t change the media and … Continued

Question 7

Can you perform transient transfection using lentiviral method? If so, how does this differ from standard transfection methods?

Lentiviral delivery involves the use of a virus carrying genetic material that integrates into cell genomes. However, there is a way to achieve transient delivery using viral vectors into cells. Using integrase-defective lentivirus, there is a specific disruption to the integration process, which inhibits integration in the cell’s genome. Therefore, after 4-5 days of culturing, … Continued

Question 8

I have read about biosafety concerns about using lentivirus. How do you address those and does that mean any specific changes for employees working in the lab?

When working with lentivirus, it is important to establish a proper biosafety lab to conduct your research. BL2 or enhanced BL2 safety labs are typically appropriate in the laboratory setting for conducting research involving lentiviral vectors. The establishment of these labs should be done according to institutional biosafety guidelines at your research laboratory. Employees should … Continued

Question 9

HEK293FT or T cells are very easily detached from cell culture vessels, several LV production protocols suggest use poly-d-lysine to coat the culture vessels. Can I avoid coating step when I use Lipofectamine 3000 to do the LV production?

Yes, you can avoid the poly-d-lysine coating step when using Lipofectamine 3000 as your transfection reagent to produce LV lentivirus. The protocol for Lipofectamine 3000 LV production requires high cell density, 95-99% confluence at the time of transfection. This prevents HEK293T (or FT) cells from detaching from the culture vessel and causing any interruption due … Continued

Question 10

Is it possible to use Lipofectamine to transfect plasmid DNA into MCF-7 cells using a 96 well method? Is there a specific protocol you could recommend?

Yes, Lipofectamine 3000 transfection reagent works well for delivery DNA into MCF-7 cells. We get ~60% transfection efficiency (GFP plasmid) by using Lipofectamine 3000 to transfect MCF-7 cells. Here are some suggestions for 96-well plate format: • The day before transfection, seed 25,000 MCF-7 cells per well of a 96-well plate. This translates to ~85% … Continued

Question 11

What concentration of virus do you recommend for infection?

This is highly dependent on the cell type that you are using for experiments. Some types of cells require higher MOI than others, and some cell types are very sensitive to high MOI’s. You need measure your LV titer, and when optimizing your experiment, try a range of MOI’s and measure protein transduction to determine … Continued

Question 12

What kind of titers can I expect in your system?

The titers of lentivirus depend on the size of your inserts or genes. Titer will decrease as the size of your insert increases. If you use our Invitrogen Lentiviral expression system: 1) pLenti6.3/V5 or pLenti7.3/V5 and ViraPower Packaging Mix 2) Lipofectamine 3000 transfection reagent for LV production protocol 3) HEK293T (HEK293T/17 ATCC) If your insert … Continued

Question 13

I have been using lentivirus, but am getting relatively low expression. Are there any specific medium requirements you recommend? Do you recommend serum? Also what do you recommend on medium changes?

We developed a Lentivirus production system using our Lipofectamine 3000 in our p-Lenti6.3/V5 (p-Lenti7.3/V5) Lentiviral Expression System. You can find the details of App Note as following website link: The LV packaging media is our specific media for Lentivirus production – Opti-MEM/GlutaMax with 5% FBS and 1X Sodium Pyruvate. It is not necessary to … Continued

Question 14

How do you address concerns about Lentivirus causing mutation of genes in the host system?

While mutations that arise from insertion are a concern with viral systems, lentiviral mutagenesis occurs at a much lower frequency making them a common choice. Most retroviruses insert randomly into the host genome, with a high incidence of insertion into the promoter/repressor regions of a gene causing disruption in the host and possible activation of … Continued