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?

Answer

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