I would like to stably express these genes in cancer cells while removing expression of wildtype protein.
There are a few ways:
Easiest way (if there is an easy way!):
1) First way requires not genome editing. generate stable cell line with shRNAmir (e.g. pGIPz vector - with puromycin resistance) against your wild type genes. Then create a recombinant construct of your gene of interest where the sequence being targeted by your pGIPz shRNAmir has been changed in the third codon for that run of sequence (i.e. so the amino acid sequence stays the same but is no longer a target as there is a mismatch in every third nucleotide). This may be in pcDNA3.1 for example and you can make stable line using Neomycin. Pro's - Reasonably fast method. Con's - Success depends on level of shRNAmir knockdown vs your over expression. shRNAmir can sometimes only give you 80% KD, which may not be enough.
More complicated way but hot at the moment:
2) CRISPR-Cas - Lots of companies getting on board with this and there are a lot of companies and or you can buy the vectors from Addgene. I won't go into the details as there are hundreds of papers and methods. But again, you can cause a small deletion on your WT gene at the genomic level. Once you have found a successful clone (which can take a while as success can be
There are a few ways:
Easiest way (if there is an easy way!):
1) First way requires not genome editing. generate stable cell line with shRNAmir (e.g. pGIPz vector - with puromycin resistance) against your wild type genes. Then create a recombinant construct of your gene of interest where the sequence being targeted by your pGIPz shRNAmir has been changed in the third codon for that run of sequence (i.e. so the amino acid sequence stays the same but is no longer a target as there is a mismatch in every third nucleotide). This may be in pcDNA3.1 for example and you can make stable line using Neomycin. Pro's - Reasonably fast method. Con's - Success depends on level of shRNAmir knockdown vs your over expression. shRNAmir can sometimes only give you 80% KD, which may not be enough.
More complicated way but hot at the moment:
2) CRISPR-Cas - Lots of companies getting on board with this and there are a lot of companies and or you can buy the vectors from Addgene. I won't go into the details as there are hundreds of papers and methods. But again, you can cause a small deletion on your WT gene at the genomic level. Once you have found a successful clone (which can take a while as success can be
Thanks very much. To follow up on your answer, I'm wondering if you have heard about the Tallen or zinc finger nucleases. Do these offer any advantages compared to the CRISPR system?
Thanks again for your response, this is very helpful.
Very well said Rob. After looking into the three systems more rigorously, I also get the sense that the CRISPR system will come to replace TALENs and zinc finger nucleases and revolutionise genome editing in the future. I'm very excited to get started: Thanks again!
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