HEK cells transfection and receptor concentration

[Laynne]

Hello!

So it started with a method of transfection with a plasmid containing the D2 receptor encoded with antibiotic resistance to gentimyocin
The cells have been maintained in the presence of gentimyocine to ensure that all cells express the D2 receptor.

However, I would like to ask whether the expression of receptors in each HEK cell are expressed at the same receptor concentration

Thank you :D

 

[skillet]

No, I've never done it but I think after a few cycles you select individual colonies, and grow them separately for a few more cycles. Then you have plates of cells originating from a single cell, so they should be identical, and you can measure the receptor concentration for each batch.

 

[Microbiologist]

Gene expression depends on many factors, like the make up of the vector (plasmid copy number, promoter, RBS) the media type the cells are cultured in, for how long and even micro-environmental influences within the media and so on. I think there would be a natural distribution "bell curve" type of variation of your exogenous D2 receptor concentration within a cell culture population. Although good culture technique like the clonal selection that Skillet mentioned will help ensure culture homogeneity and tighten up the variation.

and just because your cells are resistant to GM doesn't always mean they are expressing your D2 receptor... especially if you did the cloning yourself! Hopefully they are, but molecular biology (in my experience) can be a bitch.

 

[nuke]

Among cloned E. coli cells I sometimes get wildly different amounts of plasmid replication and later protein production despite transforming what should have been homogeneous samples with homogeneous plasmids. Not exactly sure of the reason for this. As skillet says you should always be working with an isolate from a plate that you select and then grow; I doubt cell receptor concentration is exactly the same among all cells when you are doing the culture due to the factors mentioned by microbiologist; for instance surface expression may be variable from cell to cell based on cell age from mitosis, hypoxic environment dependent on location in the culture, nutrient deficient environment, etc