TagBFP expression under MOUSE phosphoglycerate kinase promoter?

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Ali Moghimi-Khorasgani

While it is possible for a mouse PGK promoter to drive the expression of TagBFP in human LentiX293 T cells, there could be several reasons why you are not observing any signal under the microscope:

Transfection efficiency: It is possible that the transfection efficiency was low, resulting in only a small percentage of cells expressing TagBFP. You could optimize the transfection conditions, such as the amount of plasmid used or the transfection reagent used.

TagBFP expression level: Even if the transfection efficiency is high, the level of TagBFP expression may be too low to detect under the microscope. You could try increasing the amount of plasmid used or selecting cells that have integrated the plasmid.

Microscope settings: It is possible that the microscope settings are not optimized to detect the TagBFP signal. You could try adjusting the microscope settings, such as the exposure time or gain, to see if the signal becomes visible.

Cell type and culture conditions: It is also possible that the LentiX293 T cells are not the optimal cell type for expressing TagBFP or that the culture conditions are not optimal for TagBFP expression. You could try using a different cell type or optimizing the culture conditions to see if this improves TagBFP expression.

while it is possible for a mouse PGK promoter to drive the expression of TagBFP in LentiX293 T cells, there are several factors that could contribute to the lack of signal under the microscope. By optimizing the transfection conditions, increasing TagBFP expression levels, adjusting microscope settings, and/or changing the cell type or culture conditions, you may be able to detect the TagBFP signal.

Maryam Zare

Dear Ali Moghimi-Khorasgani ,

Thanks for your response.

I am sure about the transfection efficiency as I had quite good efficiency working with other plasmid backbones having a different fluorescent protein.

I have checked the brightness level of TagBFP and excitation emission wavelengths, and it seems it's on the range of the microscope I am using (although not being 100% excited, and not 100% emission can be detected); and I should be able to see some signals at least. Maybe I go ahead and check with a flow cytometer.

Best,

Maryam

Mohamed Khashan

Hi Maryam Zare

Do you think this is a normal morphology of your cells? Look like the agent you used was too toxic or you used high plasmid concentration. Try to optimize the concentration and the exposure time!

In addition, ways (Flow cytometry, FACS and fluorescence microscopy) that depend on detecting the expressed light may not express the real protein levels, so it is better to consider other procedures such WB to get better idea.

Hi Mohamed Khashan ,

In fact, the problem was with the cloning process.

I am able to see the blue signal under the microscope in the non-cloned original plasmid backbone. However, when I inserted my sequence, it might have affected the plasmid (the promoter or the TagBFP sequence). The results of sequencing will help.