hi, I'm new to cell cloning Technics.
In my case, 4t1 cell-lines was inserted IRES-vectors with one specific gene and also empty gene (for control).
As I sub cultured, I found more green cell pallets on the control vector with GFP than that of inserted gene. In addition, it can be detected with more brither GFP under florescent microscope. I believe it would be related to IRES in the vector. However, I don't know exactly how they work and affect to this phenomena.
PLEASE, Could you let me know about this?
Thanks
Hi,
I have not worked with IRES vectors and 4t1 cells yet, but I would also guess, that it has something to do with the IRES. I have found this website which compares IRES with 2A sites: https://en.vectorbuilder.com/resources/faq/should-i-use-ires-or-2a-and-which-2a-in-my-polycistronic-expression-cassette.html
I had good experience with T2A between a resistance gene and eGFP in a vector that was constructed for stable integration into CHO cells. Maybe another linker would be helpful? Eventually, when your inserted gene is downstream from the IRES, the expression is impaired (as mentioned at the website I've attached).
Hi,
in addition to Helen's helpful comments, you should be aware that your control is smaller and may be less efficiently transfected (or whatever method you use).
In addition your experimental construct is larger which likely affects transcription and or mRNA stability.
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