Question (2329)
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Confirm vector delivery: Verify successful transfection or transduction using a reporter, marker, or control vector.
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Check expression at multiple levels: Confirm expression at the RNA level (e.g., transcript detection) as well as protein level, since post-transcriptional regulation may occur.
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Consider cell type effects: CMV promoter activity can vary between cell lines and may be weak or silenced in certain cells.
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Evaluate gene-specific factors: Some genes are toxic, unstable, or tightly regulated, which can reduce detectable overexpression.
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Review culture and timing: Expression levels may depend on cell health, passage number, and time post-delivery.
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Validate construct design: Ensure the insert is in the correct orientation, reading frame, and matches the expected sequence.