I have human epithelial primary cultures. I would like to delete specific genes to reproduce certain diseases where gene mutations equals to absence of protein. Is it possible to do it with these systems at an affordable price and with an non-only-for-superexperts protocol? I know that Addgene sells some of the tools but how to use them?
Dear Clara
Of course this is possible, however time-consuming. You must have some experience in cloning and in basic cell biology to do so. We are working with CRISPr.
For this system you need basically a pair of primers to generate a dsDNA break, and a donor vector with two arms for homologous recombination within your gene. For deleting the gene you must insert somethin. I would advice you to use a resistance marker to select the positive clones by antibiotic selection. Also you can use GFP to detect positive clones, but of course you cannot selec to easily.
In conclusion, the protocol is not very expensive, but requires some time and some experience in cloning. Let me know if I can help
Best. Paco
Ok. Your explanation is easy to understand.
Would it not be the best "insertion" piece a doble gene: RFP-IRES-puromycin resistance?
In this way you will see a double validation by colour and resistance.
Is it too big? Does it have to be of the same size than the piece you are chopping away?
Clara
The insert that will delete your gene is going to be inserted by homologous recombination, so, in principle the size does not need to be similar to the piece that you are chopping away. For example we are deleting miRNA genes (100-150 bps) by inserting a puromycin resistance gene (more than 1 kb).
Best. Paco
Clara, sorry, I forgot
The only thing where size matters is the recombination segment. So, you have to design two homologous arms of about 800-1000 bps to ensure an efficient recombination after Cas9 digestion in CRISPr.
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