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 92

0.2 µ m pore size by electroelution. Immunodetection was performed with a rab-
bit anti-ER α polyclonal or a mouse anti-c-Myc monoclonal antibody (Santa Cruz
Biotechnology Inc, both used at 1:100). Transgene product was detected using
horseradish peroxidase-conjugated anti rabbit-IgG (Cell Signaling Technology,
1:2000) or anti mouse-IgG (Pierce Biotechnology Inc., 1:800) secondary anti-
bodies, as appropriate. The nitrocellulose membrane was then processed using
chemiluminescence detection reagents (Thermo scientific). The blots were then
stripped and reprobed using anti- α -tubulin (Sigma, 1:1000) to act as an internal
loading level standard. Western blot images were captured using BioRad FluorS
Imaging Unit and densitometry carried out using Scion Image software (Scion
Corporation, version Alpha 4.0.3.2, USA).
Immunocytochemistry was carried out on CTX0E03 cells fixed in a 96-well
plate (BD) using 4% PFA/4% sucrose in PBS for 15 min after 3 days growth
(control) and following 1- and 4-weeks in medium containing no growth factors
(EGF, bFGF) or 4-OHT. Anti-c-myc (1:100) and anti-ER α (1:500) were incu-
bated in 1xPBS containing 1% normal goat serum (Vector laboratories) overnight
at room temperature. After rinsing, the CTX0E03 cells were incubated for 1.5
h at room temperature using anti-mouse conjugated Alexa Fluor 488 (Invitro-
gen, 1:2000) for c-myc and anti-rabbit conjugated Alexa Fluor 568 (Invitrogen,
1:2500) for ER α . CTX0E03 cells were then counterstained by incubating with
Hoechst 33342 (Sigma, 1 µM) for 2 min. Quantification of c-myc and ER α was
carried out by manual counting of three representative fields using an Olympus
IX70 fluorescent microscope.
Authors' contributions
EM, LS, AH and JS conceived the experiments. LS, EM, RC, JH and PS designed
and performed the experiments and analyzed the data. All authors contributed to
the preparation of the written manuscript.
Acknowledgements
We thank Prof. Jack Price (King's College London, United Kingdom) for his
general support and helpful discussions. In addition, our gratitude extends to Dr.
Aaron Jeffries (King's College London, United Kingdom) for advice and guidance
on DNA methylation analysis; and to PrimerDesign for their help in develop-
ing the Alu primers. We also extend our thanks to Susan Hines (Imperial Col-
lege London, United Kingdom) and Dr. Marc Oliver Baradez (Laboratory of the
Government Chemist, LGC, United Kingdom) for their kind assistance in the