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Amplifying shRNA lentivirus

letivirus

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#1 Sharkie_green

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Posted 28 August 2015 - 04:12 AM

Hi ,

I got some lentivirus from another lab and was wondering if it is possible to amplify the virus by ourselves? and how?

 

Although the virus is replication defective, what if we cotransfect the packaging genes to  HEK cells during infection? would it work?

 

thanks in advance for helping!



#2 miST32

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Posted 17 September 2015 - 10:15 AM

If I understand you correctly, you have obtained viral particles and are asking if you can propagate them, like what is possible for adenovirus?  

This will not work with replication incompetent lentivirus because it integrates with a non-functional 5' LTR (the result of deleting the U3 region of the 3' LTR).  Hence you won't have transcription of the viral genome and cotransfecting the packaging plasmids won't do much.

Also consider that HIV is a retrovirus with an RNA genome.  It is this RNA genome that would need to be re-packaged into the new particles to generate virus.  In principle this *might* occur in a cell where all the constituent parts are available, but you'd be relying on the transfer of free RNA/unpackaged genomes into newly expressed viral envelope/packaging proteins.  I'm not an expert on this, but I would expect the resulting titer be low to none.  The level of available RNA genome in a given cell will be very low, because the copy number reflects the multiplicity of infection (MOI) used for the transduction.  In a general transduction, you're usually using an MOI of 3 or higher, depending on cell type.  The RNA genome counts per cell won't be anywhere near those produced by transfection of a plasmid with a functional 5' LTR.  You would, at maximum be limited by the amount of virus you put in... so you'd get no increase in virus over what you put in.

Virologists, feel free to correct me if I'm wrong here.

Cheers


Edited by miST32, 17 September 2015 - 10:16 AM.





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