gateway system - (Oct/25/2007 )
hi, my boss thinks about the gateway system. I know very less about it. I want to know if I made my target gene into entry vector. should I have to buy GFP, RFP expression vector or virus from invitrogen for this system? How can I switch to other company's lenti or adeno virus system?
You will need to assemble all your expression genes into the entry vector. Now you can move it into the lenti vector or any other vector of choice which supports the gateway cloning.
I have actually modified vectors (lenti & AAV) we got from other labs so that they are now gateway vectors and can be used to clone stuff into very easily after assembling everything into the entry vectors. You could use any fluorescent construct you have, you donot have to get them from invitrogen.
Invitrogen has a Vector Conversion kit that allows you to convert any vector you already have into a destination vector. Basically, you digest your vector, blunt and dephosphorylate it, pick the proper reading frame cassette (the kit comes with all 3) and do a 2-part blunt ligation. You will also need pENTR (conventional cloning) and/or pDONR (PCR cloning) vectors for getting your targets into the system.