Hey, I have been trying to transduce cardiac fibroblasts with lentiviruses. I transduced them using different concentrations of non-concentrated as well concentrated viruses. I went for as high as 100uL of the concentrated lentiviruses. But when I start selection of the transduced cells with puromycin all the cells are dead. And also the cells do not express the reporter gene (mCherry). Seeking advice on identifying and resolving the underlying issue causing these outcomes.
If you explain the time-course experimental procedures, we might be able to give you some advice.
I seed HEK293T cells at 25 to 30% confluency on Day -1. On Day 0 I transfect them for viral packaging. For all the previous experiments I changed the media after 24 hours (day 1) and collected the media after 48 (day 2) and 72 hours (day 3) of transfection. I used non-concentrated viral media and concentrated viruses both for transduction of cardiac fibroblasts. After 48 hours (2 days) in viral containing media, I changed media and replaced with the selection media. After 24 hours of selection media when I observe cells they are dead which means transduction failed.
your selection is too strong; to select for puro resistance you should use a concentration that kills the (untransduced control) cells in 1 or 2 weeks (after 4-5 changes in media) 24h is too fast
Sounds good. But the think is that I also tried to transduces the cells with reporter gene but even the reporter gene is not expressed.
Dear Waqas Ahmad
Addressing challenges in lentiviral transduction of cardiac fibroblasts involves optimizing several key factors:
■ Optimize Cell Density (generally sub confluent) and Health (prefer lower passages)
■ Viral Vector Optimization: use high titer vector with purified particles
■ Efficient Promoters: Select promoters that are effective in cardiac fibroblasts. For instance, the phosphoglycerate kinase 1 (pgk) promoter may be more suitable than the cytomegalovirus (cmv) promoter in some cell types.
■ Transfection and Transduction Conditions; use an efficient transduction enhancer
Regarding this latter point, I would recommend our LentiBlast Premium Transduction enhancer to increase viral infection and transduction.
LentiBlast Premium is a patented modified poloxamer that we developed a couple of years ago and that acts by simultaneously neutralizing electrostatic repulsions between membrane and viral particles and enhancing viral fusion with cell membrane. Due to a favorable “membrane permeable effect” limiting the transmembrane potential changes, it is non-toxic and totally compatible with cell viability.
It is a real asset to viral infection because it allows to increase the effectiveness of the viral vector, thus reducing the MOI, and consequently the immune response of the cell. Secondarily, it reduces the cost of experience. And thus I believe it would be ideal in your experiment.
LentiBlast Premium was demonstrated to be efficient of multiple cell types from Hematopoietic Stem Cells, to T lymphocytes and I am sure it would be worth give it a try for your experiment.
Some examples of publications with LentiBlast Premium:
- HEK293: Brown AL. Sci Rep . 2021 Oct 25;11(1):21008.
- Bone marrow derived macrophages: Fonseca GJ. Nat Commun. 2019 Jan 24;10(1):414.
- Hematopoietic Stem Cells: Burton OT. Immunity. 2024 Jun 11:S1074-7613(24)00277-2.
- HMEC: Nassour J. Nature . 2023 Feb;614(7949):767-773.
- Myeloid progenitor: Verdys P. EMBO J. 2024 Jul 18. doi: 10.1038/s44318-024-00173-7.
- T lymphocytes CD4+: Claireaux M. Nature Commun 2022 Jan 26;13(1):521.
- THP-1: Chirayath TW. Nat Commun. 2024 Sep 18;15(1):8179.
Would you like to give LentiBlast a try, please do not hesitate to contact me via ResearchGate or directly at: [email protected]
best regards,
Cedric
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