Is it possible to replace an existing shRNA sequence with a custom shRNA sequence into the pGIPZ vector ?
If the sequence is right, then the only way to test is through lentivirus, because the cells with which I work are hard to transfect (either siRNA transfection or lentiviral infection works but not plasmid transfection).
Appreciate for your time and effort.
There are no unique sites to replace the shRNA sequence in pGIPZ. The shRNA sequence is between XhoI and EcoRI sites but there are two EcoRI sites on the vector.
@ Wen-Yuan Hu. Yes you are right and I tried thinking a way but looks like its going to be a little complicated. I was wondering if I could use another lentiviral shRNA vector with the pGipZ packaging kit. Technically it should work because all pGipz has apart from the shRNA sequence, WPRE and psi packaging signal. So any other lentiviral shRNA vector should work if it has WPRE and psi packaging signal.
You won't be able to replace original shRNA sequence from pGIPZ.
But yes, you can use 2nd generation packaging system together with either custom made pLKO.1 plasmid carrying your shRNA or make it by yourself in pLVTHM backbone this one has very convenient MCS, but only GFP selection marker.
Best !
You can PCR amplify the 5'-mir-30 flanking + shRNA + 3'mir-30 flanking sequences and clone into another lentiviral vector. I prefer to clone into an intron before the reporter gen since it's a mir-based sequence, please note the "intron" is not referred to the HIV chimeric intron on the lentiviral vector. You can clone the mir-shRNA sequence into the 3' end of a reporter/marker gene if there is no other intron in your lentiviral expression vector. I have attached an example/figure, we clone the miRNA or mir-shRNA into the EF1a intron on the lentiviral vector.
Thank you for the suggestions. I am going to do a similar approach.
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