Some Suggestions for Systematic Gene Knockout pls - (Nov/18/2005 )
I'm a undergrad student from India. I sometimes get wacky ideas and just get stuck without anyone to say a word if its actually possible. I just read this paper on "Functional genomic analysis of C. elegans chromosome I by systematic RNA interference" published in Nature Vol 408, 16 Nov 2000. I'm really not an expert in any of these stuff, but this idea popped in my mind:
1. Take a cell line, extract the mRNA from a few cells, make the cDNA and amplify using PCR
2. Use some Enzyme to randomly cleave these cDNA fragments
3. Add plasmid DNA fragments to this soln and add an enzyme which ligates DNA. Now add exonuclease. Now, all DNA is degraded and only circular plasmid DNA remains. Also, these plasmids may or may not contain the cDNA fragments.
4. Create Viral vectors with these plasmids, and then use this solution on the cell line.
Supposing that the viral vector is such a way that it produces siRNA from the cDNA fragment encoded in it, then it should cause the repression of a large proportion of the genome, one gene in a cell, in a few cells right?
I'm askin u this because in my coll lab they're testing some molecules which induce apoptosis in an unknown way. They haven't found out the genes involved in this apoptosis induction. So, supposing we introduce this cell line we prepared above to this apoptosis inducing compound, then only the cells where the genes required for this apoptosis inducer to work, were repressed, would survive, and we SHOULD be able to find out these Genes, right?
i think it's a long and difficult way to proceed. You don't even know if your inducer acts as an inducer of protein X or destroy protein Y... There is so many ways...
As you may know, micro or macroarrays (affimetrix) exists and they can screen many genes in a quite quick way. That would be a first hypothesis.
You can then try to knockdown increased gene relevant to apoptosis mechanisms, and/or do a surexpression of relevant downregulated genes.