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cells. However, the silencing of c-mycERTAM expression observed in vivo is con-
sistent with the absence of tumor formation derived from CTX0E03 cells when
implanted in vivo [19,22].
Transgene silencing can occur at a transcriptional or post-transcriptional level.
Long-term transcriptional silencing of retrovirus-delivered genes has been shown
largely to result from methylation of the cytosine nucleotides within the promoter
regions [27]. The factors responsible for DNA methylation is reviewed in detail
elsewhere [28]. As the c-mycERTAM transgene was delivered into the CTX0E03
cell line using a retrovirus, it was reasonable to speculate that transgene silencing
had occurred through methylation of CpG islands within the transgene promoter
region. Bisulfite DNA sequencing was used to determine the methylation status
of the CMV promoter region of the c-mycERTAM transgene within CTX0E03
cells. In culture, only 6 of the total CpG sites screened were found to be methy-
lated. In the samples prepared from CTX0E03 grafted MCAo rodent brain 62
to 100% of the sites were methylated. These data unequivocally show that c-
mycERTAM expression is silenced at a transcriptional level in vivo as a result of
DNA methylation within the CMV transgene promoter region.
The c-mycERTAM technology acts at a protein rather than a transgene expres-
sion level. To mitigate the risk of the technology following intracerebral implanta-
tion of CTX0E03 cells it was important to investigate if c-mycERTAM transcrip-
tion level was a predictive measure of the recombinant protein level. Western
blot analysis of the in vivo samples proved impossible because the c-mycERTAM
protein would be too dilute to detect in whole brain extracts. In addition, IHC
analysis of the in vivo samples was impractical because of the substantial num-
ber of sections required to perform a global analysis of c-mycERTAM protein
expression. To overcome these challenges we developed and in vitro model of
c-mycERTAM silencing in CTX0E03 cells. The kinetics of transgene silencing in
vitro was slower than that observed in vivo, which provided the ideal cell culture
model to directly compare the progressive decline in c-mycERTAM transcription
with the translated protein level. Although we observed a decrease in the number
of c-mycERTAM positive cells by ICC, the relative percentage of positive cells did
not closely follow relative transgene expression levels. These ICC findings further
highlight the drawback of histological approaches because the cells were scored
positive independent of the quantity of c-mycERTAM protein present within the
cell. On the other hand, when total c-mycERTAM protein levels were measured
by western blot they were found to decrease in parallel with transgene transcript
level over the 1- to 4-week period. This data indicates that the c-mycERTAM
transcript and protein levels are closely linked in CTX0E03 cells.
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