How to generate Lentivirus? - (Oct/02/2013 )

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?

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

bob1 on Wed Oct 2 19:29:32 2013 said:

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 ?

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

bob1 on Tue Oct 8 20:57:21 2013 said:

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