c-MycERTAM Transgene Silencing in a Genetically Modified Human Neural Stem Cell Line Implanted into MCAo Rodent Brain - Genetic Engineering: Recent Developments in Applications - page 84

status of the CMV transgene promoter was investigated in CTX0E03 cells pre-
and post-implantation. The CMV promoter of the transgene contains numer-
ous potential methylation sites within a CpG rich island (Figure 3A). Bisulfite
sequencing of gDNA clones taken from control non-implanted CTX0E03 cells
showed only 2% of these CpG sites were methylated (Figure 3B). However, the
same analysis carried out on clones obtained from the CTX0E03 cell implanted
MCAo rat brain samples showed that 86.8% (SEM = 7.1) and 75.0% (SEM =
1.4) of the CpG islands were methylated at 1-week and 4-weeks, respectively
(Figure 3B). There was no statistical difference between 1- and 4-weeks, p = 0.14,
indicating that the methylation had occurred within the first week and was con-
stant to the 4-week time point. Collectively, only four gDNA transgene promoter
clones in vivo were found to be devoid of any methylated sites from a total of 99
analysed; in other words, ~96% of the transgene promoter sequences analysed in
vivo where found to contain at least one methylated CpG island.
Figure 3. In vivo analysis of CpG methylation within the c-mycERTAM promoter region in CTX0E03 cells.
CpG dinucleotide-containing regions (underlined) examined in CMV promoter of the c-mycER TAM transgene
(A). Methylation sequence analysis of the CMV transgene promoter region of in vivo and in vitro samples (B).
Ten animal grafted brains were analyzed, five at 1-week (top set) and five at 4-weeks (bottom set), in addition, a
non-implanted CTX0E03 cell sample (control). Ten clones containing the sequence depicted in (A) from each
grafted rat brain were analyzed. Each row shown represents a clone. Filled circles = methylated CpG island;
open circles = unmethylated CpG island. Percentages of global methylation are reported at the bottom of each
sample.
In Vitro Characterization
Additional experiments were carried out in vitro to determine if a reduction in
transgene transcription leads to a concomitant reduction in translated protein.