PMID- 8079511
OWN - NLM
STAT- MEDLINE
DCOM- 19941005
LR  - 20190909
IS  - 0168-1702 (Print)
IS  - 0168-1702 (Linking)
VI  - 32
IP  - 3
DP  - 1994 Jun
TI  - Studies of fowlpox virus recombination in the generation of recombinant vaccines.
PG  - 283-97
AB  - A p7.5/beta-galactosidase (7.5 lacZ) gene construct, cloned adjacent to the 
      fowlpox virus (FPV) thymidine kinase (tk) gene was used as a marker to identify 
      the products of recombination as 'blue' FPV plaques. The rFPVs were detected as 
      early as 4 h after the introduction of plasmid DNAs and by 72 h post-infection 
      (p.i.) for one transfer vector comprised 0.48% of the viral population. The 
      proportion of rFPV increased linearly from 0.073% to 0.62% as the cumulative 
      length of homologous sequences in the transfer vector increased from 0.73 to 4.5 
      kb. Two approaches using a second reporter gene, the Newcastle disease virus 
      haemagglutinin-neuraminidase (NDV HN) gene were tested to differentiate between 
      single and double cross-over events. In one, the HN gene was cloned into the FPV 
      tk gene and the 7.5 lacZ cloned outside of the homologous region. Progeny of a 
      single cross-over with FPV DNA generated an unstable plaque containing the HN 
      gene and subsequent intramolecular recombination resulted in excision of the 7.5 
      lacZ and the generation of a stable 'white' plaque. For virus grown in CEF cells 
      (tk+) in the presence of 5-bromo-deoxyuridine, only those viruses which contained 
      a tk gene disrupted by the HN gene formed plaques. This approach allowed us to 
      easily identify rFPV containing the HN gene but lacking 7.5 lacZ or other 
      bacterial sequences. In a second approach, a double cross-over between rFPV DNA 
      containing a stably expressed beta-galactosidase gene cloned into the tk gene 
      (blue plaque) and plasmid DNA containing the HN gene flanked by tk sequences 
      would allow transplacement of the 7.5 lacZ gene with the HN gene, and generating 
      a white plaque. We were unable to generate recombinant viruses with the HN gene 
      and which generated a white plaque, indicating that double cross-over events do 
      not occur at a sufficiently high frequency in FPV.
FAU - Parks, R J
AU  - Parks RJ
AD  - Department of Veterinary Microbiology and Immunology, University of Guelph, 
      Ontario, Canada.
FAU - Krell, P J
AU  - Krell PJ
FAU - Derbyshire, J B
AU  - Derbyshire JB
FAU - Nagy, E
AU  - Nagy E
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - Virus Res
JT  - Virus research
JID - 8410979
RN  - 0 (HN Protein)
RN  - 0 (Vaccines, Synthetic)
RN  - 0 (Viral Structural Proteins)
RN  - 0 (Viral Vaccines)
RN  - EC 2.7.1.21 (Thymidine Kinase)
RN  - EC 3.2.1.23 (beta-Galactosidase)
SB  - IM
MH  - Base Sequence
MH  - Cell Line
MH  - Fowlpox virus/*genetics
MH  - Genes, Reporter
MH  - Genes, Viral/genetics
MH  - Genetic Vectors/genetics
MH  - HN Protein/genetics
MH  - Molecular Sequence Data
MH  - *Recombination, Genetic
MH  - Thymidine Kinase/genetics
MH  - Transfection
MH  - Vaccines, Synthetic/*genetics
MH  - Viral Structural Proteins/genetics
MH  - Viral Vaccines/*genetics
MH  - beta-Galactosidase/genetics
EDAT- 1994/06/01 00:00
MHDA- 1994/06/01 00:01
CRDT- 1994/06/01 00:00
PHST- 1994/06/01 00:00 [pubmed]
PHST- 1994/06/01 00:01 [medline]
PHST- 1994/06/01 00:00 [entrez]
AID - 0168-1702(94)90078-7 [pii]
AID - 10.1016/0168-1702(94)90078-7 [doi]
PST - ppublish
SO  - Virus Res. 1994 Jun;32(3):283-97. doi: 10.1016/0168-1702(94)90078-7.