In the first figure (empty vector), only a short linker is in front of EGFP. EGFP is followed by IRES2 and mCherry. The cells transfected with this empty vector only express mCherry; I can not get any EGFP-positive cells via flow cytometry, which means EGFP was not expressed. Theoretically, the EGFP: Cherry ratio should be equal to 1.
However, in the second figure, I inserted the sequence of POI in front of the short linker and EGFP. This time, EGFP can be expressed.
So I am wondering if that is something wrong with my empty vector that leads to EGFP not being expressed.
Hi Yannick, since the Kozak sequence has already included the ATG start codon (up figure). Therefore, I just let the EGFP sequence start from Val (2aa). I have another empty vector_2 (bottom figure), which has an identical sequence to the one I posted, except for the linker sequence in front of EGFP. The empty vector_2 can normally express EGFP in cells.
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