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Employing Lentivirus for Efficient Transfection
We recently finished our Ask the Expert discussion on Improving lentiviral production using Lipofectamine 3000 reagent. This week we had several interesting questions focused on gene insertion, media requirements, virus concentration, titers, detached cells, biosafety, and seeding densities. There were also discussions involving transfection methods and how lentivirus compares to other methods.
During this Ask the Expert session, we also discussed how Thermo Fisher Scientific has addressed lentivirus transfection challenges with the Lipofectamine 3000 reagent. LentiVirus is extremely versatile and an efficient transfection method for 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, this versatile reagent enables high viral titers even with genes that are large or difficult to package.
This Ask the Expert session was 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.
Scientists interested in learning more about or troubleshooting lentivirus as a transfection method should visit this Ask the Expert session. Below is a sneak peek, for a full transcript of the discussion, please see – Ask the Expert – Improving lentiviral production using Lipofectamine 3000 reagent.
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.
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.
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