Difficulties in generating gene-specific Lentivirus - (Aug/09/2006 )

Hi,

I have been trying to generate Lentivirus for gene expression without much success. I used Invitrogen's ViraPower system and started from entry clone construction. However, I faced with difficulties from entry clone to the transfer to Lentiviral destination vector. The most frustration part is that after the clonase reactions, I got some clones which did not all grew in LB medium. And digestion of miniprep DNA gave confusion digestion patterns (I admit that I just used sol I, II, III for the miniprep). I tried sequencing of some miniprep DNA and the result was also not good.

I have heard that Stbl3 should be grown at 30 degree Celsius. Do you think that will help with getting positive clones with good miniprep DNA yield? I am thinking of switching to SBI system that requires direct restriction site cloning, and also because the Invitrogen reagents are very expensive.

And suggestions are very appreciated.

I am using Invitrogen's Lentiviral system.

I dont understand y u had problems tranferring from the entry vector to the lenti vector. Its a simple recombination reaction, and nearly always very effective using the clonase enzyme.

The lenti vector grows very slowly, I agree, U need to use longer incubarions.

Dont try sequencing, its difficult. I have adopted a different way to grow up maxi preps and its been working.

Ask me anything related to this vector and use of lentiviral system.

Hi,

Thanks very much for your response.

We saw some colonies after plating out the recombination-transformed bacteria and picked some to grow. Only a few however grew in the overnight culture. After we prepared the miniprep DNA from the grown bacterial clones and digested, there was no clear cut pattern. That was the reason we tried sequencing.

Did you prepare maxiprep (using Qiagen kit?) for any of your clones and were they all positive?
Should I grow the cultures at 30 degrees and incubate longer to prevent false positive and have a better yield of plasmid DNAs?

Thanks for any suggestion.

Alex

hi,

We r using Invitrogen's maxi and mini kit-bcoz its cheaper (used to have Qiagen's mini prep kit) now. if I am doing the clonase reaction (recombination), i always get 90% positive clones. I use Stbl3 cells for tranformation and works very efficiently. i agree some clones dont grow well, but its the natur eof these plasmids. I am growing all preps in 37C and it works.

If u want to make a maxi, then grow ur clone in a 10ml over the day and use it to inoculate the overnight culutre-500ml. Grow for 15 hrs. I usually get more than 1mg of DNA, sometimes upto 2mg.

For mini prep, restriciton digest, u will nearly always get a smear (unclear gel) if u use STbl3 cells as u also elute some exonuclease so DNA starts to get degraded. usually what I look for is if there is a band around the size of the insert. If I have it, then I make the maxi and the exonuclease doesnt elute from the maxi colums and one could verify the DNA again (just to be 100% sure).

I would suggest not to have mini or maxi preps growing longer than 16hrs. OFcourse for maxi u will grow the day culture and then overnigth culture - not more than 16hrs.

Good Luck !!!

Hi Scolix,

Thanks very much for the advice. We will try again using your recommendation and see how it works.
Thanks again.