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How to generate Lentivirus?

Lentivirus GFP RFP heks Stemcells

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#1 laura13

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Posted 02 October 2013 - 07:32 AM

Hi , 

 

As an immunologist, Molecular biology and cloning comes difficult to me. I am having problems generating Lentivirus. Essentially what I want to do is combine my Packaging, Envelope and GFP together in HEKs and then Infect my Stem cells so they fluoresce Green but for I can't for the life of me get past the stage where I collect a viral supernatant thats successful! I have tried with Mirus transfection reagent and with the CACL method. I am at my wits end here and I can't afford to buy in a fully made Lentivirus. 

 

Any helpful advice or information for me please?



#2 bob1

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Posted 02 October 2013 - 11:29 AM

If you can explain which steps you have performed to get to the point you are currently at, that will help us (potentially) troubleshoot.



#3 laura13

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Posted 08 October 2013 - 12:30 AM

If you can explain which steps you have performed to get to the point you are currently at, that will help us (potentially) troubleshoot.

Hi, Thanks for your reply.

 

The steps I have performed :

 

  • Midi-prepped my GFP, VSVG and VPR , got good readings and good A260:A280 ratio.
  • Then , Using Heks from a lab beside me I used Mirus TRANS-IT reagent , following their steps exactly to combine my GFP, VSV-G and VPR.
  • Placed this on HEKS.
  • Was waiting for Green, and yes they were green 
  • Put this onto Heks to see if it actually generated Lenti virus butgot nothing.

So now tried the Calcium Phosphate method with the aid of a Post doc in a different Lab , Placed on Heks , collected Supernatant which should have had functional Supernatant but nothing..... 

 

I just need to get my cells GREEN !!

 

Is this enough information ?



#4 bob1

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Posted 08 October 2013 - 12:57 PM

So you checked the integrity of the plasmids before transfecting?  If not, run some on a gel to see if they are indeed intact.  The ratios mean nothing other than that there is some DNA (or perhaps RNA there) - putting dNTPs through a spec will give you similar readings!



#5 laura13

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Posted 09 October 2013 - 01:52 AM

I

 

So you checked the integrity of the plasmids before transfecting?  If not, run some on a gel to see if they are indeed intact.  The ratios mean nothing other than that there is some DNA (or perhaps RNA there) - putting dNTPs through a spec will give you similar readings!

 

I didn't run anything on a gel .... ? Mabe that could be it then 







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