Hello
I am trying to create a stable cell line in HEK293 via Lipofectamine 3000 transfection. My plasmid is a CD63-IL10-GFP construct with Puromycin resistance. I am successful with the transfection part as the cell glow bright green after 3 days of transfection but they seem to slowly lose GFP expression after being cultured in selection media. The cells eventually no longer glow but still survive in selection media. I done a selection curve on regular cells to identify ideal dose and a kill well to make sure selection was fully complete but still having issues. Does anyone have any idea what is going on and how to fix it?
Brinley Harrington Protein expression in transfected cells can be affected by several issues, including:
1. Low transfection efficiency: Not all cells take up the DNA, resulting in low protein expression.
2. Poor vector design: Suboptimal promoter, terminator, or other regulatory elements can reduce protein expression.
3. Inefficient mRNA translation: Issues with mRNA stability, folding, or translation initiation can limit protein synthesis.
4. Protein misfolding or aggregation: Incorrect protein folding or aggregation can lead to reduced activity or proteasomal degradation.
5. Insufficient resources: Limited availability of amino acids, energy, or other resources can constrain protein expression.
6. Cellular stress responses: Activation of stress pathways can suppress protein expression or induce protein degradation.
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