Hello,
I am trying to study two mutations in a multi exonic gene. My gene is TP53 with 11 exones and I have Raji Cell line which has 2 mutation : one in Exon 6 and the other one in exone 7.
For my project, I have to do 2 different knock in in order to insert the wild type exones using electroporator.
I have been suggested to use Takara electroporation system, but this system doesnt allow me to see the efficiency of my transfection as there is no reporter gene in my construct.
I was thinking to add a GFP gene at the end of my Exon and make a ssDNA doner and insert it to my gene.
But I am afraid that inserting GFP in my gene will interfere with the protein folding and i wont be able to detect it.
Anyone has any idea how can i do it without interfering in protein folding?
If you are specifically set on adding GFP at the end of your gene, you can include a self-cleaving element such as a P2A between your gene and the GFP (if you do this be sure to remove the STOP codon in your gene). Another option is to include an internal ribosome entry site (IRES) between your gene and the GFP (in this case leave the STOP codon in your gene). Both of these strategies should allow for bicistronic expression of your gene and the GFP and not interfere with protein folding.
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