Did anyone infect EO771.lmb with lentivirus?

09 January 2023 1 2K Report

I am trying to infect EO771.lmb cells with lentivirus to express luciferase and Tdtomato. My cells are not expressing anything. I tried spinning the virus for 90 min with media+polybrene, but it did not work.

If anyone has any ideas, please let me know. Thank you

Mohammad Alzeyadi

hello ..

It sounds like you're experiencing some difficulty getting your EO771.lmb cells to express luciferase and Tdtomato after infection with your lentivirus. Here are a few potential explanations for why your cells may not be expressing the reporter proteins:

Transduction efficiency: The transduction efficiency of the virus may be low, resulting in few cells being infected and expressing the reporter proteins. Factors that can affect transduction efficiency include the multiplicity of infection (MOI), the presence of polybrene, and the health and confluency of the target cells.

Cell culture conditions: The culture conditions of the EO771.lmb cells might not be optimal for the virus to infect and express the reporter proteins. it's best to check that the cells are in a log phase and in high density culture and also make sure that the cells are in good condition.

Virus production: The virus might not be produced in high titer or active, it could be that the virus is not stable, not concentrated enough, or even damaged in some way.

Viral integration: It's possible that the virus is integrating into the genome of the EO771.lmb cells, but the integrated copies of the reporter genes are not being expressed. This could be due to problems with the viral promoter or issues with the target cell's transcriptional machinery.

Plasmid construction: the construct of the reporter genes might not work properly in these cells and the reporter genes might not be expressed.

Selectable markers: EO771.lmb cells are resistant to puromycin, if you are using a puromycin resistance cassette in the plasmid to select the transduced cells, this could be the problem as the cells might not be able to express the genes.

One thing you could try is optimizing the multiplicity of infection, increasing the amount of polybrene in the infection media, or trying a different transduction enhancer. You can also try to concentrate the virus by ultracentrifugation, tangential flow filtration, or size-exclusion methods.

It's important to check your results using multiple approach

Regards

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