In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.
Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.
Thomas
J L
JL
Laboratoire de Biologie Cellulaire, INRA, CNRS UMR106, Université Claude Bernard, Villeurbanne, France.
Afanassieff
M
M
Cosset
F L
FL
Molina
R M
RM
Ronfort
C
C
Drynda
A
A
Legras
C
C
Chebloune
Y
Y
Nigon
V M
VM
Verdier
G
G
D000818:Animals; D030801:Animals, Genetically Modified; D001354:Avian Leukosis Virus / Q000235:genetics*; D015139:Blotting, Southern; D002642:Chick Embryo; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D007763:Lac Operon; D008845:Microinjections; D012190:Retroviridae / Q000235:genetics*; D001616:beta-Galactosidase / Q000032:analysis
D001616:beta-Galactosidase