I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

The issue you're encountering with the THP-1 cells expressing GFP but not the exogenous gene could be due to several factors:

Firstly, consider transcriptional silencing or promoter interference. Even though GFP and the exogenous gene are under the same promoter, variations in chromatin structure or epigenetic modifications could inhibit the transcription of the exogenous gene while allowing GFP expression. This can occur due to the integration site of the lentiviral construct in the host genome, where integration into a transcriptionally inactive region or one marked by repressive chromatin could prevent the exogenous gene's expression [1][2].

Secondly, the viral titer and multiplicity of infection (MOI) could play a significant role. An insufficient or uneven viral load could result in a mixed population where only a subset of cells receive a functional copy of the lentiviral construct capable of expressing both genes. This could be exacerbated by the use of the same batch of virus for two rounds of infection, possibly leading to variability in infection efficiency [3][4].

Thirdly, post-transcriptional regulatory mechanisms such as RNA stability, differential splicing, or RNA interference might differentially affect the exogenous gene compared to GFP. This could lead to varying expression levels even though both genes are under the same promoter [5].

To address these issues, verify the integration sites of the lentiviral construct using methods like PCR or sequencing. Increasing the MOI and ensuring even distribution of the virus can improve infection efficiency. Additionally, checking for epigenetic modifications and performing single-cell analysis might help elucidate the variability in gene expression among the infected cells [1][2][3][4][5].

Reference

[1] Seok, H., Jeon, J. H., choi, H. K., Choi, W. S., Park, D. W., Wi, Y. M., ko, K. S., & Peck, K. R. (2019). 611. Fosfomycin Resistance of Multidrug-Resistant Escherichia coli and Mechanisms of Fosfomycin Resistance. Open Forum Infectious Diseases, 6, S285 - S285.

[2] González-Zorn, B., Teshager, T., Casas, M., Porrero, M., Moreno, M., Courvalin, P., & Domínguez, L. (2005). armA and Aminoglycoside Resistance in Escherichia coli. Emerging Infectious Diseases, 11, 954 - 956.

[3] Ali, M. R. (2023). Phylogenetic Groups and Antibiotic Resistance Characteristics of Uropathogenic Escherichia coli. Journal of Communicable Diseases.

[4] Hong, T., Moland, E. S., Abdalhamid, B., Hanson, N., Wang, J., Sloan, C., Fabian, D., Farajallah, A., Levine, J., & Thomson, K. (2005). Escherichia coli: development of carbapenem resistance during therapy.. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 40 10, e84-6 .

[5] Mark, L., Sigmund, C., & Morgan, E. A. (1983). Spectinomycin resistance due to a mutation in an rRNA operon of Escherichia coli. Journal of Bacteriology, 155, 989 - 994.

Park Sowon

Thank you so much for the response and your interest in this matter. I need to clarify that I didn't infect the previously transduced cells again, I mean, I performed the transduction procedure twice on seperate cells but the source of lentivirus was the same (I had packaged lentivirus just once and alequted that and strored them in -80). since, this construct had worked for my lab mates, so we are sure that the construct and the integration site are correct. I am trying to make new viruses and transduce again. I am wondering what if some mutations had happened during reverese transcriptase function during the virus proliferation. Does it make sense?

I had used the higest MOI and I think I distributed viruses evenly but thanks for mentioning that, I will be more careful about this next time.

your two genes being expressed with the same promoter (and maybe the same mRNA=(with a T2A sequence?) I would rather suggest that you have some recombination in your plasmid or during reverse transduction by the lentivirus. do you know if you have cells expressing your GOI but no GFP? I would rather make clones and check the expression of the GOI and keep those that express the two proteins. trying to have 100% of cells expressing both could be difficult and take time.

Didier Poncet

Thanks for your response. I know that the promoter is the same for both GFP and GOI. we don't have the T2A sequence in the construct and mRNAs are probably seperated (I didn't design the construct, I have to check it). Currently, I am just checking the expression of GOI as external receptors by HA staining, and based on the FACS data, I don't have cells just expressing the GOI without GFP. So, one issue could be the problem in transferring of the receptors to the cell surface. But overall, since my colleague could get almost 98 pecent of cells expressing both GFP and GOI with the same plasmid, I am expected to get the same result. However, another previous lab member with the same plasmid had reported the same result as mine.

I appreciate again your time and your guidance in this matter.