Biology Reference
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CTX0E03 Cell Detection and Quantification in Vivo
Four weeks after the MCAo, a total of 450,000 CTX0E03 cells were implanted
bilaterally into the putamen. The rats were sacrificed 1-week or 4-weeks post-
implantation, their brains were removed and sections encompassing the injection
tracts were dissected and snap frozen in liquid nitrogen. The Alu and c-mycER TAM
assays were then carried out on the gDNA and cDNA isolated from each tissue
section, respectively. CTX0E03 cells were detected in all implanted animals at
both time points, with a range of 28,151 to 179,184 CTX0E03 cells detected
per brain. The total number of CTX0E03 cells detected, per rat brain, is shown
at 1-week (Figure 2A) and 4-weeks (Figure 2B) post-implantation. An aliquot of
the CTX0E03 cell suspension prepared for implantation was retained and used to
determine the pre-implantation c-mycER TAM transcript copy number. The average
c-mycER TAM transcription level for the three CTX0E03 cell suspensions prepared
in this study was found to be 215.7 (SEM = 13.2) copies per cell (Figure 2C). This
is compared to the population of CTX0E03 cells detected in vivo, where the av-
erage number of c-mycER TAM transcript copies per CTX0E03 cell was calculated
to be 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks (Figure 2C).
The number of CTX0E03 cells, the number c-mycERTAM transcripts calculated
per tissue section and the resultant number of c-mycERTAM transcript copies
per CTX0E03 cell, are shown in Table 1. c-mycER TAM transcripts were detected
within approximately 50% of the tissue sections containing CTX0E03 cells at
both time points. It is important to note, in no instance was a c-mycER TAM tran-
script signal detected where CTX0E03 cells were absent.
Figure 2. Detection of CTX0E03 cells and c-mycER TAM transcription in grafted rat brain. Total cells found at
1-week (A) and 4-weeks (B) post-implantation by Alu qPCR. Animal numbers 103, 304, 401, 402 and 603
were vehicle injected only control brain samples. Absolute quantification of c-mycER TAM transcript level per
CTX0E03 cell, in cell suspensions prior to implantation (control) and in vivo (C).
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