transfecting cells with viral particles

Giovanni Parisi via methods%40net.bio.net (by gparisi from farmbiol.uniba.it)
Tue May 6 03:39:33 EST 2008


hi all,
I am trying to transfect some fibroblasts and HEK 293 using viruses I 
produced that transiently express shRNA. This is a tecnique never used 
in my laboratory so I have not methods to tritate viruses so I am using 
different volumes of obtained viruses. The first silencing experiment 
was done using viral particles obtained from SIGMA and I had some 
silencing but last time I had not silencing with viruses particles I 
produced. Someone told me that producing viral particles with mission 
system from SIGMA using HEK 293T cells is a very efficent process. Is 
it possible that am I using a low concentration of viruses?
The second question is the following: I have intention to modify 
lentiviral vector pLKO1 replaceing U6 promoter with CMV promoter and 
cloning  GFP cDNA in way to transfect cells to control efficiency of 
transfection. Is it possible to do it?
thanx



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