The researchers developed a novel non-viral single doxycycline inducible system that can efficiently regulate gene expression in stem cells both in vitro and in vivo. They generated stable clonal cell lines from mouse neuroblastoma cells (N2a) and human embryonic stem cells (hNSC-ESC) that responded to doxycycline by eliminating GFP expression within 3-8 days. GFP expression was restored 9-10 days after doxycycline removal. When transplanted hNSC-ESCs labeled with quantum dots were subjected to doxycycline in mice, GFP expression was reduced by 61-56% compared to controls. This suggests the system can control gene expression in stem cells without using viral vectors

1. Myungsik Yoo, Christopher Park, Jennifer Moore, Melitta Schachner
W. M. Keck Center for Collaborative Neuroscience, Department of Cell Biology and Neurosciences, Rutgers University, Piscataway, NJ
Non-viral single doxycycline inducible system for human neural stem cell-derived
embryonic stem cells
1. Abstract
2. Introduction
3. Methodology
- Plasmid vector construction
The vector used for the initial construct was a pd2EGFP (Clontech) back bone,
subcloned with Chicken actin promoters (CAG) driving GFP and transactivator,
oppositely, and seven repeat tetracycline response elements (7XTRE) in between
the two CAG promotors.
- Predifferentiation of hNSC-ESC
We followed a slightly modified adherent monolayer differentiation protocol from
human embryonic stem cells, first published by Austin Smith’s lab (Nat, 2007)
- Transfection protocol with Amaxa to generate pTet-off GFP-hNSC-ESC
The transfection of proliferated pre-differentiated cells was done using the Amaxa
96 well transfection kit (Lonza, Amaxa)
- Clonal stable selection after transfection: N2a, hNSC-ESC
After 7 and 14 days, selection of N2a and hNSC-ESC clonal cells, respectively,
were picked into a new dish by G418 (200 mg/ml).
- Cell transplantation into cyclosporin immunosuppressed spinal cord
Cell transplantation (105 cells/ml) was performed immediately after opening of
spinal cord. 1 µL of the cells were transplanted at the center of spinal cord with
the injection lasting for 7 minutes.
Embryonic stem cell derivatives are potential avenues of therapy to cure
irreversible neuronal cell damage (Abdel-Salam 2011). In order to more efficiently
use embryonic stem cell derivatives, we hypothesized using an inducible
paradigm. Initially, the inducible system was developed in eukaryotic cells and
organisms using antibiotic and hormone regulating systems for clinical and basic
research, these were started in 1995 using chrimeric transactivator (tTA) fused
bacterial Tet repressor with viral protein 16 (VP-16) (Gossen et al. 1995).
Advanced binary tetR-mediated inducible gene regulation system, applicable to
eukaryotic cells had better regulation than the initial system by providing less
leaking and less toxicity (Yao et al. 1998). More recently, the lentiviral vectors were
developed for gene delivery to primary cells and non-immortalized cells (Cockrell
et al. 2007). However two major issues arised; the lenti-viral system could not
package the big-sized target cDNA well because of its limited size packaging and
the virus DNA sequences in the plasmid vector are silenced by the virus protection
mechanism in the cells (Suzuki et al. 2006). Thus, we have developed a novel
non-viral single doxycycline inducible system, which works efficiently in
immortalized neuroblastoma cells (N2a) by doxycycline and predifferentiated
hNSC-ESC. The clonal stable human cell line pTet-off GFP-hNSC-ESC also works
efficiently In vivo in the cyclosporin immunosuppressed spinal cord.
5. Conclusion
7. References
8. Funding
We have successfully constructed a non-viral single inducible system, which
efficiently regulates in immortalized neuroblastoma cells (N2a) and
predifferentiated hNSC-ESC. In addition, we found the system’s capability to
function in vivo testing, using GFP-hNSC cells line in the spinal cord.
These results suggest that the pTet-off GFP system can be delivered in a non-
viral manner and can be used to control the expression of doxycycline inducible
genes in stem cells in vitro and in vivo.
6. Discussion
New Jersey Commission for Spinal Cord Research
-Abdel-Salam OM. (2011). Stem cell therapy for Alzheimer's disease. CNS Neurol. Disord.
Drug. Targets 10: 459-485.
-Cockrell AS, Kafri T. (2007). Gene delivery by lentivirus vectors. Mol. Biotechnol. 36: 184-
204.
-Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H. (1995). Transcriptional
activation by tetracyclines in mammalian cells. Science 268: 1766-1769.
-Nat R, Nilbratt M, Narkilahti S, Winblad B, Hovatta O, Nordberg A. (2007). Neurogenic
neuroepithelial and radial glial cells generated from six human embryonic stem cell lines
in serum-free suspension and adherent cultures. Glia 55: 385-399.
-Suzuki M, Kasai K, Saeki Y. (2006). Plasmid DNA sequences present in conventional
herpes simplex virus amplicon vectors cause rapid transgene silencing by forming
inactive chromatin. J. Virol. 80: 3293-3300.
-Yao F, Svensjo T, Winkler T, Lu M, Eriksson C, Eriksson E. (1998). Tetracycline repressor,
tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives,
regulates inducible gene expression in mammalian cells. Hum. Gene. Ther. 9: 1939-1950.
Figure 2. Plasmid vector map of non-viral single Tet-off GFP
system for doxycycline-dependant transgene regulation. The
vector, initially, used for the construct was a pd2EGFP (Clontech)
back bone, which was subcloned using multi cloning sites. The
genes, Chicken actin promoter (CAG), highly active in human
neural stem cells, which drives the GFP gene, and transactivator
gene, were separately constructed and subcloned to the final
vector oppositely. The PCR product of seven repeat tetracycline
response elements was subcloned between the two CAG
promotors. The benefits of this system are the use of a non-viral
sequence, single plasmid system driving two separate strong CAG
promoters, multiple tetracycline response elements, and a strong
repressor Kruppel-associated box fused with a tetracycline
repressor to enhance inducible.
A B C
A B
C
100
50
D
%cellviability
0 0.5 2
mg/ml dox
Figure 7. Doxycycline was non-toxic and did not affect GFP
expression in the non-inducible human neural GFP stem cell
line. Non-inducible human stable cell line generated after
transfected pCAG-GFP as the same protocol with inducible human
cell line. Cells without doxycycline (A), cells with 0.5 mg/ml
doxycycline (B), and cells with 2 mg/ml doxycycline (C). Cells
exposed with doxycycline (B and C) showed no difference in cell
number and viability to the cells lacking exposure to doxycycline
(D). Scale bars, 100 µm.
Figure 6. GFP off, time-dependently regulated using pTet-off GFP stable
clonal cell line human neural-stem cells (hNSC), by doxycycline.
Illustrated in A,B, and C, the cells exposed to doxycycline resulted in the
decrease of GFP. D, E, F illustrate cells in the absence of doxycycline,
resulting in the increase of GFP. Cells without doxycycline after 4 days (A),
cells with doxycycline 1 mg/ml after 4 days (B), and cells with doxycycline 1
mg/ml after 8 days (C). Scale bars, 100 µm
Figure 5. Pre-differentiated human H9 embryonic stem cells express
neural and neuronal markers. A) A phase contrast image of the cells
including rosette forming units. B, C, and D, illustrate immunofluorescence
staining of A2B5, Nestin, and doublecortin (DCX). Bar graph of cells positive
with immunoreactivity, most cells being positive for neural stem cell marker
nestin, about 20% of cells positive for glial progenitor marker A2B5, 15% of
cells positive for early neuronal progenitor marker DCX, however astrocyte
marker GFAP and embryonic stem cell marker Oct4 were not detected (H).
Scale bars, 100 µm.
Figure 4. GFP on and off, time-dependently regulated using
pTet-off GFP stable cell line N2a-#12 by doxycycline. Illustrated
in A,B, and C, the cells exposed to doxycycline resulted in the
decrease of GFP. D, E, F, and G illustrates cells in the absence of
doxycycline, resulting in the increase of green fluorescent protein.
Cells without doxycycline after 1 day (A), cells with doxycycline 1
mg/ml after 3 days (B), and cells with doxycycline 1 mg/ml after 6
days (C). Cells without doxycycline after 1 day (D), cells without
doxycycline after 3 days (E), cells without doxycycline after 6 days
(F), and cells without doxycycline after 9 days (G). Bar graph of
GFP on and off (H). Scale bars, 100 µm.
Figure 3. Generation of single clonal Tet-off GFP N2a stable cell lines.
After transfection of the Tet-off-GFP plasmid, the cell lines were selected by
G418 (200 mg/ml) for 7 days. Individual clonal cell lines were picked and
separately passaged. Cell lines N2a-#5 (A,D), N2a-#12 (B,E), N2a-#13 (C,F),
were tested without doxycycline (A, B,C) and with 1 mg/ml doxycycline (D, E,
F), for 5 days. The cell line N2a-#12 was the best cell line regulated by
doxycycline. The bar graph (G) marks the percentage of GFP present for
each cell line with and without doxycycline. Asterisks indicate significant
differences between the groups **p<0.01 assessed by one-way ANOVA
followed by Tukey’s post-hoc analysis. Error bars represent SEM. Scale bars,
100 µm.
Figure 1. Schematic representation of the mechanism for ptet-off-GFP
system using doxycycline. This system uses the single plasmid
doxycycline Tet-off system in which seven repeated tetracycline response
elements are located between two chicken actin promoters which drive
expression of GFP and the tetracycline reverse transactivator oppositely. (a)
In the absence of doxycycline, the tetracycline response elements are latent
to the two Chicken actin promotors, which drive expression of the green
fluorescence protein gene and the transactivator. (b) In the presence of
doxycycline, TREs recruit transactivators fused with a strong repressor
Kruppel-associated box, which leads to the silencing of two gene expression
through the inactivation of CAG promotors.
Figure 8. Non-viral single Tet-off hNSCs-GFP regulated by doxycycline, in vivo. The spinal cords were
transplanted with 10^5 cells in cyclosporin-immunosuppressed mice, and 7 days after transplantion the mice were
perfused and analyzed. The mice which were not fed doxycycline in drinking water, showed similar levels of GFP
and red quantum dot expression after 7 days (A). The mice which were fed doxycycline in drinking water, showed a
decrease of GFP expression while the quantum dot showed no significant change, after 7 days (B). The bar graph
(C), illustrates the comparison of the mice fed with doxycycline and the mice fed without doxycycline. In 7 days the
cells expressing GFP were reduced by about 61% and in 10 days the cells expressing GFP were reduced by about
56%. Asterisks indicate significant differences between the groups *p<0.05 as assessed by one-way ANOVA
followed by Tukey’s post-hoc analysis. Error bars represent SEM. Arrows (in A-B) indicate the injury site. Scale
bars, 0.3 mm
4. Results
The conventional approach for the doxycycline inducible system typically utilizes a
lentiviral delivery system to target the cell of interest. However, a significant
disadvantage of lentiviral delivery is the difficulty faced when trying to efficiently
package viral constructs that are larger than 10kb. We have successfully
developed a novel non-viral system that can express genes in response to
doxycycline in vivo and in vitro. After clonally isolating a stable mouse
neuroblastoma cell line (N2a) and the H9 human embryonic stem cell line (hNSC-
ESC), we were able to demonstrate that 0.5 mg/ml doxycyline completely
eliminated GFP expression after 3 days in N2a cells and 8 days in hNSC-ESC.
GFP expression was restored 9 days after doxycycline removal in N2a cells and
10 days after removal in hNSC-ESC. To test the ability of these cells to respond to
doxycycline in vivo, we transplanted 105 hNSC-ESCs labeled with quantum dots
(red; 655 nm) into the non-lesioned spinal cord. When the mice were subjected to
doxycycline, sacrificed and analyzed for the ratio of quantum dot expressing cells
to GFP expressing cells, we found that GFP expression was reduced by 61% and
56% in the doxycycline treated group. These results suggest that the pTet-off GFP
system can be delivered in a non-viral manner and can be used to control the
expression of doxycycline inducible genes in stem cells in vitro and in vivo.
The non-viral inducible single system used here was capable of efficiently
regulating (over 98%) in vitro conditions for immortalized neuroblastoma cells
(N2a) and pre-differentiated hNSC-ESC. However, in vivo results for hNSC-ESC
showed an estimate of 68% regulation.
The position of the tetracycline response elements (TREs) proves to be significant
for expression. The position of TREs must be placed at 5’ head in order to function
properly, if placed at 3’ end then expression will be inhibited, regardless of the
presence of doxycycline.