4.2.1.1 - Infection of RCAS-p in vitro
4.2.1.1.1 - Radioactive in situ hybridisation
To check the spread of the viral construct in vitro and that the hRAR-p probes were
species-specific, CEF(O) were plated out on to glass slides and then infected with either sense or antisense RCAS-p viral supernatants. Dr. A. Rowe then probed these with either a 35s-labelled antisense RNA probe specific for hRAR-p transcripts or with a ^^S-
The sense transfected cells hybridised with the antisense probe, showed labelling (see figure 4.1a). As a control they were also hybridised with the sense probe, no labelling was observed (see figure 4.1b). In contrast hRAR-|5a showed no labelling with the
antisense probe (figure 4.2a), but did with the sense probe (figure 4.2b).
4.2.1.2 - Infection of RCAS(BP)-|3 in vivo
4.2.1.2.1 - Microinjection of RCAS(BP)-P into the developing chick embryo
The RCAS (BP) constructs were used to infect chick embryos in vivo, as this vector has been shown to spread more efficiently in vitro than RCAS.
The titers of the concentrated virus stocks were :
RCAS (BP) hRAR-p sense 4.4 - 6 .8 x 10^ viral particles per ml.
RCAS (BP) hRAR-p antisense 5.4 x 10^ - 1.8 x 10^ viral particles per ml.
Virus was injected into the developing right wing primordia of chick embryos ranging from stages 7+ through to 16. Figure 4.3 shows a fate map of the developing wing, which was used to determine where to inject the viral supernatant (Chaube, 1959). The embryos were operated on at an early stage to obtain good viral spread. The embryos
were harvested between 2 and 5 days after manipulation. Previous work using this vector has shown that the entire limb bud should be positive for viral spread after 48 hours (Morgan et al, 1992). After harvesting the embryos, they were fixed in 4% paraformaldehyde and then wholemount in situ hybridisation to hRAR-P transcripts was
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■i 0* Figure 4.1Radioactive in situ hybridisation on cells infected with RCAS-fs a) hybridised with an antisense RNA probe
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Figure 4.2Radioactive in situ hybridisation on cells infected with RCAS-(3a a) hybridised with an antisense RNA probe
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Figure 4.3
Outline o f su ccessive em bryonic stages 7-13 (A -H ) and stage 17-18 (I), with the prospective wing and leg regions (the upper two rows o f dots represent the wing regions and the lower two rows the leg regions). The centre o f the node is indicated by (X). The solid horizontal line by the side o f the marks in each o f the diagrams represents the approximate lateral extent o f the wing area in each o f the stages. Arrow in C indicates rapid expansion o f intermediate flank material.
Of 148 embryos injected with the sense construct, 42 survived. All were analysed by wholemount in situ hybridisation with a species-specific human RAR-p antisense probe. 34 had no detectable hRAR-p transcripts. 8/42 showed limited spread of the virus from the site of injection. Figures 4.4 and 4.5 show distribution of hRAR-p transcripts within
the embryo. Some injections were carried out directly into the neural tube. When these embryos were analysed for viral spread (as above) no hRAR-P transcripts were
observed in any of the cases. Table 4.1 summaries the results of injections with the sense construct.
39 injections with antisense viral supernatant were also carried out to act as controls. 14/39 survived and were analysed for viral spread. The same problem was found as with
the sense injections, that the viral construct was not spreading within the limb bud (see
table 4.2 for summary of the antisense injections). Due to the lack of success we had with the injections, we decided upon a different approach to express hRAR-p ectopically
in the developing chick wing.
4.2.1.2.2 - Grafts
A pellet of transfected RCAS(BP)-ps cells was grafted into the right wing bud. Prior to
grafting, a parallel culture of cells was stained with the monoclonal antibody 3C2 to gag
to ensure that the cells were 100% positive for the virus. The idea was that the graft would be continuously producing virus from each cell, thus acting as a localised source of virus enabling the entire limb to be infected. This was not the case. When the grafts were harvested and analysed for hRAR-p transcripts, all showed very patchy
expression, a very small proportion of cells were infected. It would have been expected that if not all the cells within the limb bud were positive for hRAR-p transcripts then at
least a high proportion of cells should be. Table 4.3 is a summary of the grafting experiments.
Distribution of hRAR-P transcripts in the embryo after injection with RCAS(BP)-ps at stages
8+ and 11+.
a) Infection of leg, 4 days post infection, stage 28 Injected at stage 11+, with concentrated supernatant Scale bar = 300 |im.
b) Infection of wing bud, dorsal surface, 4 days post infection, stage 27 Injected at stage 8+, with unconcentrated supernatant
Scale bar = 200 ^im.
c) Symmetrical expression of hRAR-p transcripts in the somites, 4 days post infection, stage 28
Injected at stage 11+, with concentrated supernatant Scale bar = 300 |im.
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b)
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b)
Figure 4.5
Distribution o f hRAR-p transcripts in the embryo after injection with R C A S(B P)-ps at stage 14.
a) Infection o f the ventral surface o f a chick leg, 5 days post infection, stage 31 Injected at stage 14, with concentrated supernatant
b) Infection o f the dorsal surface o f a chick leg, 5 days post infection, stage 31 Injected at stage 14, with concentrated supernatant