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 80

In this study, we developed a novel and sensitive method that is able to analyze
absolute gene expression within human genetically modified cells grafted into
rodent brain. This method used the human specific Alu genomic sequence to
detect and quantify CTX0E03 cells grafted within the rodent brain background
[25,26]. Absolute quantification of c-mycERTAM transcription made it possible
to calculate the number of c-mycERTAM transcript copies per CTX0E03 cell.
Using this methodology, c-mycERTAM transgene silencing was shown follow-
ing grafting of CTX0E03 cells into MCAo rat brain. Although not intentionally
incorporated into the design technology of c-mycERTAM, silencing of the CMV
transgene promoter in vivo demonstrates an additional safety feature of this tech-
nology and of the CTX0E03 cells.
results
Alu and c-mycertAM Assay design
The analysis of the c-mycER TAM transcript expression per CTX0E03 cell, both in
vivo and in vitro, was dependent upon the ability to sequentially purify genomic
DNA (gDNA) and RNA from the same biological sample using the All DNA/
RNA Kit (Qiagen). The gDNA preparation yielded one single band by agarose
gel electrophoresis with no evidence of degradation or contaminating RNA (Fig-
ure 1A). The RNA preparation yielded the expected 28S and 18S bands, again
with no degradation or DNA contamination visible (Figure 1B). Standard curves
were then generated to determine: 1) the number of CTX0E03 cells present in a
gDNA sample by Alu qPCR (Figure 1C); and 2) the number of c-mycER TAM tran-
scripts present in a retro-transcribed RNA reaction sample (Figure 1D). The Alu
sequence assay could reproducibly detect human CTX0E03 gDNA with as few
as one CTX0E03 cell per reaction; whereas, the c-mycER TAM assay could detect as
few as 10 copies per reaction.
Validation of Alu and c-mycERTAM Assays
The Alu sequence and c-mycER TAM transcription assays were developed in the
presence of rat brain gDNA or cDNA, as described in the Methods. These experi-
ments involved mixing purified CTX0E03 DNA/cDNA with purified rat brain
DNA/cDNA. However, in order to verify that CTX0E03 cells grafted in rat brain
could be processed and analysed effectively together a positive control experiment
was carried out. This experiment consisted of harvesting and processing the rat
brain tissue immediately after a 6 µ l injection of CTX0E03 cells was delivered.
It was determined by counting using a haemocytometer that the cell suspension