We do this routinely in our laboratory to test mutations in BHV-1gB. We
use a gB null BHV-1 virus maintained on a gB-expressing cell line. By
electroporating non-expressing cells with linear plasmid containing the gB
orf (it's very important it be linear) then superinfecting with the null
virus we rescue the null virus by homologous recombination. This only
works if the mutations in the gB gene are non-lethal. In this case the
gene is running off it's original promoter. When we inserted BHV-1gB gene
into HSV-1 it ran at 10% of normal efficiency with the BHV-1 promoter and
100% efficiency using the HSV-1 TK promoter. It will depend on the virus
you're using.
There is considerable interest in using herpesviruses to express foreign
antigens e.g. Kit et al. 1991 in Vaccine 9:564-572. The large number of
"redundant" genes allows fairly easy insertion of foreign DNA. We replaced
the thymidine kinase gene of HSV-1 with glycoprotein B of BHV-1 to produce
a pseudodiploid HSV. The wide tissue ranges of most herpesviruses also
make them desirable vectors.
The constructs we have made in HSV-1 and BHV-1 have been stable. That is
if the original recombinatin reformed the genome correctly. In a couple of
cases there was genome rearrangment and these were not stable. Again,
this would depend on the size of insert, location and specific
herpesvirus.
Selecting for the recombinant virus is the trick. The gB null BHV-1 virus
was developed by Thomas Mettenleiter and co-workers and has the Lac
inserted into the gB gene. You can identify recombinants by white/green
plaques using the gB expressing cell line. We used a non-expressing cell
line thus selecting for rescued virus. In the HSV-1 pseudodiploid
experiments we inactivated the TK gene. A TK-negative virus can be
selected for by using bromovinyl deoxyuridine, a nucleotide analogue.
Unfortunatly, this reduces the size of plaques, since the BHV-1gB
insertions had inactivated UL24 this also results in small plaques. It
gets a bit difficult to see anything in the monolayer. But it does work.
Earl
------------------------------------------------------------------------------
Earl Linwood Blewett Veterinary Microbiology, WCVM || Slow Swimmer
University of Saskatchewan Saskatoon, Saskatchewan S7J 2R5 || Slow Cyclist
E-mail blewette at herald.usask.ca || Slow Runner
Phone (306) 343-6772 || "TriSloth"
On 26 Oct 1994, Zafer Yildirim wrote:
>> I know that , for example , vaccinia viruses have been used in
> transfer of foreign genes into cells. The gene is incorporated
> into the viral genome by homologous recombination from a plasmid
> if I am not wrong. Has anybody tried this or any other method to
> incorporate genes into the herpes virus genome ? Which part of
> the viral genome is targeted in general ? How stable would it
> be (I mean the recombinant virus) ? What promoters for expression
> of the foreign gene would be used ? How can we select for the
> recombinant virus? ...
>> Thanks in advance.
>>>
