The virus works by three RNA transcripts being spliced from the provirus (the full sequence encoding, gag pol env and hRAR-P) after it has been integrated into the host
cell's DNA. These are then transported to the cytosol where they are translated. Due to the nature of the virus, it will spread from cell to cell as well as being passed on to daughter cells with the host cell's DNA, see chapter 3 (Stryer, 1981). It has been shown by staining with a monoclonal antibody, 3C2, to gag that the virus is entering the cell, transcribing and the primary transcript is being translated into protein. The gag products are cytoplasmic proteins. As it has been proved that this product is present, the vector itself is being integrated into the host CEF.
The second transcript, encoding env, is also being spliced as shown in the northern blot. This transcript is important as the envelope protein is host specific and if it were not functioning, the virus could not enter the cell. The reason that the hRAR-P transcript is
sequences of the insert. As will be discussed in chapter 5, the reason is most likely to be
due to the sequences of the insert than to do with the vector. It is possible that the some rearrangement has occurred within the hRAR-p insert or that the insert is not compatible with the vector. The data from the northern blots indicates that the hRAR-P transcripts
observed with the radioactive in situs corresponds to transcripts I and II, which both contain hRAR-P but not to transcript III which should eventually translate the functional
protein.
The results of the immunocytochemistry did not conclusively prove whether protein was being translated. The immunocytochemistry appeared to be cross reacting with endogenous protein, as all the cells, including the antisense cells, had background staining and no higher staining was observed in the sense transfected cells. The western blot also showed non specific bands. The expected hRAR-p band of 50,000Mr was not present in the sense cells, indicating that hRAR-p protein was not being produced from
the viral construct.
In the CAT assay experiment, if the sense transfected cells had been producing functional RAR-p protein, an increase in the amount of CAT activity would have been expected. Eager and colleagues (1991) transfected cells with both hRAR-p and cRXR
and the (TRE3)3-r/: -CAT construct and treated them with RA. They found that both cRXR and hRAR-p transactivated gene expression in response to RA, and that the
response was greater with hRAR-p. In this assay I found that the RCAS(BP) Ps
transfected cells did not transactivate gene expression in response to RA, when compared to the controls. This result indicates that the RCAS(BP)-ps transfected cells were not producing hRAR-P protein, hence no increase was seen in the amount of CAT
activity.
The results of the RA binding assay showed no evidence for hRAR-p protein being present in the RCAS(BP) Ps transfected cells. No increase in the number of RARs was
detected. The aim of this assay was to try to ascertain if functional RAR-p was being
produced from the cells transfected with RCAS(BP)-Ps. The control used was cells
transfected with the antisense construct which should not produce any more RARs. The
RA binds to the receptors in the cell and by adding a 2000 fold excess of unlabelled RA to half the cells at each concentration of RA, the amount of background labelling can be eliminated from the calculations. The reason for carrying out the assay is that it
would be expected that the sense cells would show an increase in the number of RARs due to ectopic receptors being produced by the transfected cells and that from this it could be concluded that functional protein was being produced, unfortunately this was not the case. As can be seen from the results of this assay, the sense cells appear to contain the same number of RARs as the controls. This assay does not provide a clear
answer as to whether the cells are producing an increase in receptors, nor does it show that the cells are not producing excess numbers of receptors. This assay was carried out several times and each time no clear deduction was able to be made. The sense transfected cells always showed approximately the same number of receptors as the controls or less, therefore either the viral construct is not functioning correctly or this assay is not sensitive enough to detect an increase in the number of RARs that are being produced above background levels. It is more likely that the problem lies with the viral construct and the inserted hRAR-P sequences, as in a similar assay when a cDNA-
deduced hRAR protein was inserted into a eukaryotic expression vector pSG l under the control of an SV40 promoter and transfected into HeLa cells and a retinoic acid carried out, the hRAR showed high affinity for retinoic acid in its binding capacity. A higher level of binding was observed in the transfected cells than in the controls (Petkovich etal, 1987). This was not observed in the RCAS(BP)-p assays.
The virus spread very poorly in vivo and it seems that transcripts of hRAR-p are being
transcribed, but as shown by the northern the transcript required to translate the message to protein is present in very low abundance. This seems to correlate with the functional
assay results that if protein is being produced it is not in high enough levels to be detected above endogenous levels.