6 replies to this topic

[dandan]

I have got a lentivirus plasmid with my target gene expressed in it ( the lentivirus vecotr is already recombinanted in HEK293 cells). Now i wanna transduct this lentivirus plasmid into my cells. i have amplified this lentivirus plasmid in the bacteria, purified, and transfected into the cells by lipid. the target gene is Flag tagged, i ran the WB, blotted with anti-flag antibody but barely got anything. i have no experience on virus cloning, is anybody have ever done this could give me some suggestion, is it right to do like what i have said?  
thanks a looooooot.

[lab_member]

I guess you mean you cloned your target gene into a lentiviral vector and transfected the plasmid into the cells by lipid methods (and NOT by lentiviral particles).

I am not sure whether you have used any selection or not but I have also used a lentiviral vector for chemical transfection and due to its larger size (~7kb) compared to other overexpression vectors (~5kb) the transfection efficiency is rather low (it think only about 20% or even less).  Therefore, I think it is not surprise that only very little cells are expressing your target gene hence you only get very little... i really regret using a lentiviral vector since i did not actually package it into a lentivirus...

I also heard some of my lab people mentioned in overexpression is that there may be some frameshift problem hence not producing the protein you want but i think the possiblity is very low if you are careful...i am not sure because i never did any overexpression before...

Hope it helps

[dandan]

dandan, on Mar 28 2009, 04:30 AM, said:

I have got a lentivirus plasmid with my target gene expressed in it ( the lentivirus vecotr is already recombinanted in HEK293 cells). Now i wanna transduct this lentivirus plasmid into my cells. i have amplified this lentivirus plasmid in the bacteria, purified, and transfected into the cells by lipid. the target gene is Flag tagged, i ran the WB, blotted with anti-flag antibody but barely got anything. i have no experience on virus cloning, is anybody have ever done this could give me some suggestion, is it right to do like what i have said?  
thanks a looooooot.

yes, i got a lentivirus plasmid from a collaborator's lab, i think it is just a tranfer vector, so if i wanna transduct to the cells i need to package it in the 293 cells, but we dont have the protection to allow us to carry out the transduction. i just transfect the plasmid into my host cells directly. but the vector is huge, 11kb, even bigger than yours, i could barely detect anything by the marker, so i dont know, maybe i need to ligase onto other vectors. i have no idea how could those plasmid work in my collaborator's lab.

Edited by dandan, 02 April 2009 - 04:10 AM.

[Functional Screens]

In general people clone cDNA or shRNA into lentiviral vectors and then prepare lentiviral stock by transfecting the lenti vector with Gag-Pol and VSVg vectors into 293T cells.  It's a BioSafty Level 2(+) setup, you should be able to do it in your lab.  This is the way to generate stable lines or to deal with hardly transfected cells
For highly transfectable cells like 293T, transient transfection should be fine.  Therefore, people use smaller vectors such as pcDNA.  In this case, you can also generate stable lines (if you want) by transfecting linerized expression vector and following by drug selection.

[dandan]

Functional Screens, on Apr 6 2009, 05:54 PM, said:

In general people clone cDNA or shRNA into lentiviral vectors and then prepare lentiviral stock by transfecting the lenti vector with Gag-Pol and VSVg vectors into 293T cells.  It's a BioSafty Level 2(+) setup, you should be able to do it in your lab.  This is the way to generate stable lines or to deal with hardly transfected cells
For highly transfectable cells like 293T, transient transfection should be fine.  Therefore, people use smaller vectors such as pcDNA.  In this case, you can also generate stable lines (if you want) by transfecting linerized expression vector and following by drug selection.

thank you very much for you explaination. so that means it is safe to do the package on the lab bench, and i also could ligase my cDNA onto a smaller vector to do transient transfection, is that right?

[Functional Screens]

dandan, on Apr 7 2009, 12:49 AM, said:

Functional Screens, on Apr 6 2009, 05:54 PM, said:

In general people clone cDNA or shRNA into lentiviral vectors and then prepare lentiviral stock by transfecting the lenti vector with Gag-Pol and VSVg vectors into 293T cells.  It's a BioSafty Level 2(+) setup, you should be able to do it in your lab.  This is the way to generate stable lines or to deal with hardly transfected cells
For highly transfectable cells like 293T, transient transfection should be fine.  Therefore, people use smaller vectors such as pcDNA.  In this case, you can also generate stable lines (if you want) by transfecting linerized expression vector and following by drug selection.

thank you very much for you explaination. so that means it is safe to do the package on the lab bench, and i also could ligase my cDNA onto a smaller vector to do transient transfection, is that right?

Yes, you can transiently express your gene of interest by transfection, also packaging lentivirus for long-term expression.

The information of Biosafety in Microbiological and Biomedical Laboratories (BMBL) can be
downloaded from the following link: http://www.cdc.gov/o...5th_Edition.pdf

[dandan]

Functional Screens, on Apr 7 2009, 05:08 PM, said:

dandan, on Apr 7 2009, 12:49 AM, said:

Functional Screens, on Apr 6 2009, 05:54 PM, said:

In general people clone cDNA or shRNA into lentiviral vectors and then prepare lentiviral stock by transfecting the lenti vector with Gag-Pol and VSVg vectors into 293T cells.  It's a BioSafty Level 2(+) setup, you should be able to do it in your lab.  This is the way to generate stable lines or to deal with hardly transfected cells
For highly transfectable cells like 293T, transient transfection should be fine.  Therefore, people use smaller vectors such as pcDNA.  In this case, you can also generate stable lines (if you want) by transfecting linerized expression vector and following by drug selection.

thank you very much for you explaination. so that means it is safe to do the package on the lab bench, and i also could ligase my cDNA onto a smaller vector to do transient transfection, is that right?

Yes, you can transiently express your gene of interest by transfection, also packaging lentivirus for long-term expression.

The information of Biosafety in Microbiological and Biomedical Laboratories (BMBL) can be
downloaded from the following link: http://www.cdc.gov/o...5th_Edition.pdf

got it, thanks a lot