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 88

conclusion
In conclusion, we have demonstrated that c-mycER TAM transgene expression levels
in CTX0E03 cells were significantly reduced in vitro, upon growth factor and
4-OHT withdrawal, and in vivo following implantation. We demonstrated that
in vivo transgene silencing occurred through DNA methylation. In addition, the
in vitro results showed that c-mycER TAM transcription level was representative of
the c-mycER TAM protein level. The data demonstrate that uncontrolled in vivo cell
growth arising from c-mycER TAM technology is unlikely based on both the silenc-
ing of the c-mycER TAM gene expression reported here and the specific requirement
for the 4-OHT ligand to activate the c-mycER TAM protein. Furthermore, these
data demonstrate that the retroviral insertion of stable transgene constructs into
human neural stem cells with the CMV promoter element is not an inherently
unsafe procedure for the production of cellular therapies.
Methods
ethics statement
Fetal tissue used to derive CTX0E03 cells was obtained in accordance with na-
tionally (UK and/or USA) approved ethical and legal guidelines. All animal work
was performed in accordance with the UK Animals (Scientific Procedures) Act
(1986) and approved by the local animal Ethical Review Committee.
derivation of ctX0e03
The CTX0E03 clonal cell line was derived from human fetal brain cortical tissue
of 12 weeks' gestation, as described previously [19]. Briefly, a mixed culture was
initiated from the dissociated cortex tissue and was infected with an amphotro-
pic replication-incompetent retroviral vector (pLNCX-2; Clontech) encoding the
c-mycER TAM transgene. The CTX0E03 clonal cell line was isolated using a ring
cloning method. The CTX0E03 cells were routinely cultured as a monolayer on
mouse laminin (Trevigen, AMS Biotechnology, UK) freshly coated flasks in se-
rum free medium (RMM) containing the growth factor supplements 10 ng/ml
bFGF (Peprotech), 20 ng/ml EGF (Peprotech), and 100 nM 4-OHT (Sigma). All
experiments were carried out using CTX0E03 cells between passage number 33
to 37, which corresponds to a population doubling level (PDL) ranging between
65 to 75.