I have a plasmid encoding my gene of interest which has GC rich DNA sequence , which fully sequenced only in presence of 10% DMSO. This plasmid was transfected in HEK293 cells . After selection by G418, some cell colonies were over expressing the a truncated form of the protein
I changed the vector by sub cloning and the problem happened again what could be the reason for that and how it should be fixed ?
thank you
Yasser S. Hassan
Hello Wedad, one possibility would be a nonsense mutation that you can fix by optimizing your codon for the HEK293 cells.
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Dominique Liger
Hi there,
The presence of a protease cleavage site and/or the fact that part of the protein is disordered (ie. more susceptible to degradation) could explain such result.
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