Question on methods for transduction and transfection - (Nov/14/2006 )
Hi, I'm currently working on a project which explores gene therapy vehicles. Basically, I am trying to formulate a new vehicle to transfer the transgene into the cancer cells efficiently. My professor has indicated he wants a proposed mechanism for the production of this vector and I have very limited knowledge in this field (aside from a Cell Biology course with some mention of biotechnology). So, I came up with this procedure after some research but I am not sure if this works or even makes sense.
The new vehicle: Retrovirus carrying the transgene is inserted into CIK cells which are then injected into human body/cancer site.
1) Forming the plasmid which contains the therapeutic gene + selection markers.
2) Inserting plasmid into the retrovirus.
3) Generate titre of the retrovirus from 2).
4) Insert retrovirus into CIK cells and culture.
5) Select CIK cells which contains the retrovirus.
6) Culture CIK cells containing the retrovirus.
I need details on how are these techniques done and also the various methods available for each step. I have found some on the internet but I do not know the feasibility/compatability of each method for my case.
For example, I am not sure if using GFP or antibiotics to test for successful transfection/transduction is better. Or, if there are other methods which can do this.
One big question: Do I check the success of the transduction and transfection at each step (after step 2 and also at step 5) ? Or can I just check once at step 2 (for plasmid into virus) or once at step 5?
Also, I am trying to find the protocol for inserting the recombinant plasmid into a retrovirus, but have no luck in that.
After reading through the other posts which may help in my situation, I think I am not ready to work on this alone as I have never heard of some terms used. I know I am asking a lot in one post, but I hope you will be able to help in any part as it all adds up. I would greatly appreciate that.
1) regarding the commercial plasmid maps available, i would suggest to have a look at them to see the important features.
as you'll do retroviral vector, check for retroviral ones (for ex)
the plasmid should contain some packaging genes important for retrovirus production and selection marker for bacterias (ampicilin is more confortable, but see cayla for more antibiotics) and for mammalian cells
2 ) the retrovirus is produced in packaging cell line (like 293PACK cells) : you need to transfect these cells and the retrovirus is in culture supernatant.
3 ) see 2
4 ) see sequabrene or infection procedures on cultured cells
5 and 6 ) just culture with antibiotic