Both use lentiviral vector backbone plasmids and sleeping beauty transposons for a gene transfer experiment. Cells (NK and T cells) express the eGFP protein and survive through antibiotic selection but can not detect the gene expression of interest. Interestingly, we use the same promotor (EF1alpha) to express both the gene of interest and eGFP (eGFP located after the GOI in the plasmid design). I already tried the same plasmids in the HEK293T cell for confirmation, as they are efficiently transfected and able to detect the GOI by WB and flow cytometric analysis. Any idea, suggestion, or experience regarding this issue is appreciated.
There are several potential reasons why you might experience successful transfection but no expression of the gene of interest (GOI) in your gene transfer experiment using lentiviral vector backbone plasmids and sleeping beauty transposons. Some possible explanations include:
It may be helpful to perform additional experiments or analyses to try to identify the cause of the issue. For example, you could try using a different promotor to drive the expression of the GOI, or you could try analyzing the mRNA levels of the GOI to see if it is being transcribed at normal levels. It may also be helpful to consult with a researcher with expertise in gene expression and regulation to get additional insights and suggestions.
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