Sponsored by: ThermoFisher Scientific
Session ends: August 21st, 2015, 3:00pm MST
Answers by: Xin Yu Thermo Fisher Scientific, Inc.
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.
Please take advantage of the opportunity to ask our expert a question and participate in a lively discussion on Improving LentiVirus production.
Questions & Answers
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 […]» Read MoreWe 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 […]» Read MoreThe 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 […]» Read MoreThis 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 […]» Read MoreYes, 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% […]» Read MoreYes, 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 […]» Read MoreWhen 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 […]» Read MoreLentiviral 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, […]» Read MoreNo, 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 […]» Read MoreFor 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 […]» Read MoreWe 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 […]» Read MoreYes, 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 […]» Read MoreUnlike 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.» Read MoreLipofectamine 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 […]» Read More