Questions on Baculovirus system

[Bart_Corsaro_at_USLRMG01 at INTERNETMAIL.PR.CYANAMID.COM]

 >1-  Maximum how many extra bases I can have before the ATG 
>codon of my "gene"? 

In the Baculovirus system the late promoter has the sequence of "ATAAG" with 
transcription initiating usually from the T or second A.  The optimum distance 
between this sequence and the start codon appears to be approximately 70 bp, 
but the distance does vary between 3 and greater than 100 bps.  The maximum 
amount of untranslated leader DNA that you can insert will really be dependent 
on the transfer vector that you choose; however, I wouldn't count on the 
distance being greater than 100 bp.  


 >2-  It appears that I do not have a lot of desirable restriction 
>sites around the beginning of the ORF, how am I going to get rid of the 
>extra bases?

To get rid of the unwanted leader DNA you could try nested deletions with 
ExoIII/Mung Bean Nuclease Kits.  These usually work well but be prepered to 
screen alot of clones to find one with the proper size insert.

> 3-  I am planning to use polyhedrin promoter based vectors. The 
>kits that the companies sell are very expensive (almost $500 for 5 
>transfections). Which one is the best choice? Is occ based selection too 
>difficult?

While I haven't used them, I have heard the the kits from Pharmingen usually 
work quite well.  The Baculogold kit is suppose to be very good.  It initially 
got bad reviews because of some poor quality control (uncut viral DNA which 
gave very high background of nonrecombinants).  I believe that these problems 
have been corrected.  If you use a selection with occlusion minus, it is very 
east to detect in a plaque assay.


 Good Luck

Bart Corsaro
Lederle-Praxis Biologicals
West Henrietta, NY
EMail: Bart_Corsaro_at_USLRMG01 at internetmail.pr.cyanamid.com