baculovirus kit opinions needed

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Mon Mar 2 13:13:36 EST 1998


At 12:47 2/25/98 -0500, VoiD wrote:

Kyle:

I am by no means an expert.  However, I have had some experience with
baculovirus systems.

>Hello, I am looking for opinions of various baculovirus cloning kits 
>available, to determine the answers to the following questions:
>1. How easy is it for a beginner in insect cell culture to get up and 
>running in this system?

In my view, if you can do normal tissue culture, then you'll likely be fine
as the same techniques apply.  You will have to commit an incubator to 23'C
and possibly a incubator with a rotating platform.

>2. Which kit gives the highest percentage of recombinant virus?

Hard to say. I've read a number of articles (JI and JIM) that use
Pharmigens BaculoGold.  A colleage of mine uses this system and has had
great results.

>3. Which kit has the simplest protocol for generation of high titre virus?

That depends on who you ask!   I once thought the Gibco Bac to Bac would be
the easiest and although Gregg Wells (a participant in this group) had good
luck with this system, it was a veritable nightmare for my lab.   I was
enticed with the small size of the vectors (~4.4 kb) and the use of 3
antibiotics and ITPG to coherse the transfer vector to recombine with the
bacmid, generating the DNA to infect Sf9 cells.  We had major problems
making sure the bacmid actually had our DNA of interest and in most cases
it didn't despite the color selection.  

Basically I've had to shut down the projects involved as we were spending
too much time (6 months) trying to de-bug the system.  Now, I'm just
searching for the system wherein I can still do the projects but not have
to remake all my DNA inserts for the Ni fusion based vectors.  At the
moment, I'm strongly leaning toward the BaculoGold from Pharmigen where if
there is no insert, the baculoviral DNA has  a lethal gene.  Ideally, only
what has been recombined with your DNA in the MCS will be viable. 

>I know the Invitrogen BaculoGold system touts itself as producing 99% 
>recombinant virus, but is this system suitable for the novice?

So does Pharmigen's.

I once used the old BlueBacIII from InVitrogen and it worked well.
However, at that time  (1992) a 10.5 kb transfer vector was too big for any
large cDNA as we had trouble getting a 5.5 kb insert in.  Recombined virus
wasn't lethal, so we spent 6 weeks and 4 rounds of blue/white screening
before we got what we wanted.

Try a medline of "baculovirus expression" vs a few journal names and you
might just find what people have been using with success...

Hope this helps,
David
=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
Never take life seriously. Nobody gets out alive anyway.
=============================



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