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.

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 the gene size 4Kb: you will need 1.2ug p-Lenti expression vector +1.8ug ViraPower™mix
The total DNA amount is 3ug for each transfection reaction using a 6-well plate format.

You can find details of the protocol at the following website link: https://www.lifetechnologies.com/content/dam/LifeTech/global/life-sciences/CellCultureandTransfection/pdfs/Lipofectamine3000-LentiVirus-AppNote-Global-FHR.pdf

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.

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, 1.2x10^6 cells per-well are needed for forward transfection versus 3.6x10^6 cells for reverse.

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 cells. However, we have not done a comparison for Lentiviral production.
Infection the target cells with lentivirus, the cell culture media can be in presence or absence pep/strep antibiotic.

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 delivery is often associated with genomic integration which can be avoided with other transfection methods.

An alternative method for transfection that is highly effective in non-dividing and primary cells is the use of mRNA and MessengerMAX. This is a fast and easy method using a non-viral delivery system that provides high efficiency transfection.

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 use crude lentiviral cell supernatant, there may be a slight increase in GFP positivity that could translate into a 1 fold higher titer. Therefore, you would just need adjust the MOI to infect your target cells.

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, gene expression will be gone. When deliverying a gene using standard transfection, the introduced nucleic acid (mRNA for transient delivery) will only be present in the cell for a limited amount of time before getting degraded, and will not integrate into the genome.

Both methods have the similar effect, but different concept. Transient transfection by lentiviral method has more advantages than standard transfection. It has high gene delivery efficiency to non-dividing cells, like primary, neuronal cells, blood T cells , B cells and hard transfection cells which standard transfection method has very low delivery efficiency.

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 be trained in how to handle and appropriately discard waste products and byproducts generated when using lentiviral vectors.

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 to adding DNA/reagent complex, changing media, harvest LV by replacing fresh media.

It is still important to carefully handle the cells when adding complexes and changing media. It is recommended that any addition should be done towards the side of the culture vessel, and also use pre-warmed media.

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% confluence at the time transfection
• Next Morning, transfect the cells (for each transfection reaction):

a) Tube 1: Dilute 100ng DNA ( high quality, endotoxin-free) + 0.2ul of P3000 reagent in 5ul Opti-MEM I
b) Tube 2: Dilute 0.3ul Lipofectamine 3000 reagent in 5ul Opti-MEM I
c) Combine the two tubes and incubate at room temperature for 5 minutes to form transfection complex
d) Following the complex formation step, add 10ul complex directly to the cell culture medium in each well.
e) 6 hours post transfection, change cell media and replace with fresh MCF-7 culture medium
f) Post-tfx 48hrs, harvest the cells

The medium change at 6 hours post transfection is not required when using Lipofectamine 3000, but if the cells are sensitive, such as the case with MCF-7, medium changing will help to reduce the cell death.

ATCC MCF-7 cells growth rate is low. We use Gibco DMEM, high glucose, GlutaMax Supplement, pyruvate + 10%FBS to culture the MCF-7. The supplement sodium pyruvate will help MCF-7 growth a lot.

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 your optimal level for your specific experimental set up. To increase transduction efficiency, you can add 8ug/ml of Polybrene, associated with a 30 minute room temperature spin (2000 rpm) to increase transduction efficiency.

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 is: 5Kb, expect a crude LV titer: 0.1-9.0 x10^6 TU/ml

Note: Reference titers are based on antibiotic selection or %GFP measurement in HT1080 cell line.

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: https://www.lifetechnologies.com/content/dam/LifeTech/global/life-sciences/CellCultureandTransfection/pdfs/Lipofectamine3000-LentiVirus-AppNote-Global-FHR.pdf

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 change media post-transfection if you are planning to concentrate the lentiviral particles 24-48hrs post-transfection (with two supernatant harvests), or you can use benzonase treatment to eliminate the remaining DNA plasmids. However, if you are going to use crude lentivirus in your next step to transduce your target cells, the remaining DNA/reagent complex might be toxic to your cells, so changing the medium at post-infecting 6hrs is recommended.

Important to keep in mind, use crude lentiviral cell supernatant which had remained DNA/reagent complex to measure the viral titer using the %GFP method, there may be a slight increase in GFP positivity that could translate to a 1 fold higher titer. In this case, you would need to adjust your MOIs to account for the perceived increase when setting up your experiment.

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 oncogenes. However, several studies have indicated that this is not a big problem when using Lentivirus, a subset of retroviruses which has led to their utilization in clinical applications such as gene therapy.

If potential insertional mutagenesis is a concern for your experimental set up, consider using an mRNA transfection approach, which has been shown to be effective in hard to transfect cell lines, and usually why lentiviral expression systems are used. While expression will be transient, Lipofectamine MessengerMax reagent may provide you with an alternative to lentiviral infection.

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