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Xin-Yu
Qin, Kun-Tang Shen, Department of General Surgery, Zhongshan
Hospital, Fudan University, Shanghai 200032, China
Xin Zhang, Zhi-Hong Cheng, Xiang-Ru Xu, Ze-Guang Han,
Functional Genomics Division, Chinese National Human Genome Center
At Shanghai,Shanghai 201203, China
Supported by the “Hundred Talents” Program of Shanghai
Municipal Government, No.98BR018
Correspondence to: Prof. Xin-Yu Qin, Department of General
Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032,
China. xyqin@zshospital.com
Telephone: +86-21-64041900 Ext 2663 Fax: +86-21-64038472
Received 2001-09-14 Accepted 2001-12-08
Abstract
AIM: Artificial
β-cell lines may offer an abundant source of cells for the
treatment of type Ⅰ
diabetes, but insulin secretion in β-cells is tightly regulated
in physiological conditions. The Tet-On system is a “gene
switch” system, which can induce gene expression by administration
of tetracycline (Tet) derivatives such as doxcycline (Dox). Using
this system, we established 293 cells to an artificial cell line
secreting insulin in response to stimulation by Dox.
METHODS:
The
mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by
the polymerase chain reaction (PCR), and was inserted downstream
from the promoter on the expression vector pTRE2, to construct a
recombined expression vector pTRE2mINS. The promoter on pTRE2
consists of the tetracycline-response element and the CMV minimal
promoter and is thus activated by the reverse
tetracycline-controlled transactivator (rtTA) when Dox is
administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin
were co-transfected in the tet293 cells, which express rtTA stably.
Following hygromycin screening, the survived cells expressing
insulin were selected and enriched. Dox was used to control the
expression of insulin in these cells. At the levels of mRNA and
protein, the regulating effect of Dox in culture medium on the
expression of proinsulin gene was estimated respectively with
Northern blot, RT-PCR, and radioimmunoassay.
RESULTS:
From
the 28 hygromycin-resistant cell strains, we selected one cell
strain (tet293/Ins6) secreting insulin not only automatically, but
in response to stimulation by Dox. The amount on insulin secretion
was dependent on the Dox dose (0,10,100,200,400,800 and 1000μg·L-1),
the level of insulin secreted by the cells treated with Dox (1000μg.L-1
) was 241.0pU·d-1 ·cell-1 , which was
25-fold that of 9.7pU·d-1 ·cell-1 without
Dox treatment. Northern blot analyses and RT-PCR further confirmed
that the transcription of insulin gene had already been up-regulated
after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also
induced in a dose-dependent manner. However, the concentration of
insulin in the media did not increase significantly until 5 hours
following the addition of Dox.
CONCLUSION:
Human
proinsulin gene was transfected successfully and expressed
efficiently in 293 cells, and the expression was modulated by
tetracycline and its derivatives, improving the accuracy, safety,
and reliability of gene therapy, suggesting that conditional
establishment of artificial β-cells may be a useful approach to
develop cellular therapy for diabetes mellitus.
Qin
XY, Shen KT, Zhang X, Cheng ZH, Xu XR, Han ZG. Establishment of an
artificial β-cell line expressing insulin under the control of
doxycycline.World J Gastroenterol 2002;8(2):367-370
INTRODUCTION
Owing to the steady improvement in methodology for islet and whole
pancreas transplantation over the past three decades, this approach
remains as the only means which is likely to substitute completely
the function of the impaired β-cells in typeⅠdiabetics
patients[1].However, the limited availability of human
donors, the immunological rejection, the low survival percentage of
grafts, and the high expense limit the broad-scale applicability of
islet transplantation[2-4]. As a result, in recent years
much effort is being made to improve insulin secretion in the
patient's non-beta cells to avoid the immune destruction[5-9].
To successfully mimic beta cell function, it is necessary to
overcome three major obstacles: proinsulin synthesis, proinsulin
processing, and mature insulin storage and regulated secretion. Our
previous study (unpublished) and studies by other authors had
demonstrated that correct proinsulin processing and insulin storage
can be obtained in somatic cells transduced with the mutated
proinsulin gene[10-13], so the major problem to ectopic
insulin expression is the difficulty of reconstructing in non-beta
cells the highly regulated insulin secretion of the normal beta
cells[14-19] .In 1992, Gossen and Bugard[20]
developed the tetracycline (or doxycycline)-induced gene expression
(Tet-on) system, which is useful for controlling the expression of
targeted genes in quantitative manner and for determining the roles
of the gene products in cellular functions[21-26]. In the
present study, using this system, we generated the first non-beta
cell line with deoxycycline-regulated insulin secretion. The
strategy described here will contribute to the development of the
use of insulin gene therapy for patients with typeⅠdiabetes.
MATERIALS
AND METHODS
Tet-on system
pTet-on regulator plasmid (pTet-on),pTRE2 response plasmid (pTRE2)
and pTK-Hyg plasmid, which contain hygromycin-resistance gene, were
purchased from Clontech. Using the polymerase chain reaction, the
proinsulin cDNA was amplified from!expression vector pcDNA3.1/C-mINS,
which had been reconstructed from plasmids pcDNA3.1/C and
pN-PEPCK-INS (the genomic sequence of human!proinsulin, kindly
provided by Professor LI Changchen, Dalian Medical University) in
our lab. The primers used in the amplification created restriction
sites Bam HI and Hind Ⅲ
which allowed to clone the human proinsulin cDNA into the expression
vector pTRE2. The sequences of the forward and reverse promers were:
5'-cat ggatcc tgccatggccctgtggatg-3 and 5'-cag aagctt
gcaggctgcgtctagttgc-3' respectively. After verification by
restriction and sequencing, the vector, pTRE2.mINS, for expressing
human insulin was generated.
Cell
culture and gene transfection
To simplify the transfection procedure, the tet-293 cell line (human
embryonal cell), which had been transfected with the plasmid pTet-on
and stably expressed reverse tetracycline-controlled transactivator
(rtTA), was purchased from Clontech. So the first step of
transfection and clone selection was left out. The culture medium
was DMEM supplemented with 100mL·L-1 fetal bovine serum
and G418 (100mg·L-1 ). The tet-293 cells were maintained
at 37℃
under a 50mL·L-1 CO2 atmosphere until the
cells were 80% confluent, then pTRE2.mINS was cotransfected with
pTK-Hyg in a ratio of 20:1, using Lipofectin reagent (Gibco-Brl).
After selection in the culture medium containing 30mg·L-1
hygromycin (Sigma) for more than eight weeks, one clone
(tet-293/Ins6) expressing insulin under the control of Dox was
selected by screening from 28 clones.
Radioimmunoassay
Tet293/Ins6 cells (1.0×106) were seeded in
35-mm-diameter dishes, and treated with Dox at 0,10, 100, 200, 400,
800 and 1000μg·L-1 for 24h, or at 1000μg·L-1
for 0, 15 and 30min, 1, 2.5, 5, 12 and 24h. The amounts of insulin
secreted in the medium were measured with specific radioimmunoassay
kit (Haikerei Co. Peking).
RT-PCR
Total RNA was extracted from cells with TRIzol (Gibco), and the
first chain of cDNA was prepared by RT with SuperscriptIIreverse
transcriptase (Gibco) using a oligo(dT)11 primer. The proinsulin
gene was amplified with Taq polymerase (Progema). The PCR primers
for proinsulin gene were: sense 5'-tgccatggccctgtggatg-3',and
anti-sense 5'-gcaggctgcgtctagttgc-3'.
Northern
blot analysis
For each Northern blot, 10μg total RNA was loaded per lane and
separated, transferred onto nylon membrane (Schleicher and Schuell,
Germany) and crossed-linked by UV irradiation. The hybridization
probe specific for proinsulin, was labeled with (32P)dCTP
using a DNA labeling kit (Takera). Following hybridization at 68℃
for 2h, blots were washed twice for 30min at room temperature with
solution 1 (2×SSC,0.5g·L-1 SDS) and solution 2 (0.1×SSC,1g·L-1SDS),
respectively, and exposed to a phosphorimaging screen overnight. The
blots were then washed with 5g·L-1 SDS at 95℃
until complete removal of the probe and rehybridized with human
β-actin cDNA (Clontech).
RESULTS
Establishment of a stable cell line excreting insulin controlled by
Dox
After screening in the culture medium containing hygromycin for more
than eight weeks, a total of 28 independent Hyg-resistant cell lines
were created from the tet-293 cells which had been cotransfected
with pTR2.mINS and pTK-Hyg. Of the 28 clones, one (tet-293/Ins6) was
obviously regulated to secrete insulin by Dox. The amount of insulin
in the medium secreted by the cells treated with Dox (1mg·L-1
) was 241.0pU·d-1 ·cell-1 , which was
25-fold that of 9.7pU·d-1 ·cell-1 released
by the cells without Dox treatment.
Verify
the regulation of Dox on insulin secretion in tet-293/Ins6 cells at
the protein level
Tet-293/Ins6 cells were amplified and passaged (1.0×106
cells) into 16 35-mm dishes, and the concentrations of insulin in
the culture media were measured with RIA. As shown in Figure 1, the
level of insulin secretion increased from 18.2 to 241.0pU·d-1
·cell-1 over a range of Dox concentrations from 10μg·L-1
to 1000μg·L-1 . Only a slightly increasing tendency
of insulin concentration was observed at 2.5h following the addition
of 1 mg·L-1 Dox to the culture medium. However, the
amount of insulin increased significantly after exposing the cells
to Dox for more than 5h (Figure 2).
Figure 1(PDF)Insulin
secretion from tet-293/Ins6 cells after treatment with Dox at
different concentrations for 24h.
Figure 2(PDF)Time
course of insulin secretion from tet-293/Ins6 cells after activation
with Dox at 1000μg·L-1 in 24h.
Figure 3
The
conditional transcription of insulin gene by Dox in the tet-293/Ins6
cells using β-actin as loading control.
Verify
the regulation of Dox on insulin expression in tet-293/Ins6 cells at
the transcriptional level
Theoretically, the Tet-on system exerts its regulatory effect on
target genes at the transcriptional level. So RT-PCR and Northern
blot methods were used to further demonstrate the inducibility of
insulin by Dox. After tet-293/Ins6 cells were cultured in the media
containing different concentrations of Dox for 24h, it was found
that 10μg·L-1 Dox could induce insulin expression
obviously, and that the insulin mRNA level was up-regulated
dramatically by Dox in a dose-dependent manner (Figure 3A). As shown
in Figure 3B, the result of RT-PCR indicated that the transcription
of insulin gene ha d already been up-regulated significantly after
exposing tet-293/Ins6 cells to 1mg·L-1 of Dox for 15
minutes.
DISCUSSION
Insulin-dependent diabetes mellitus (IDDM) results from the
autoimmune destruction of the insulin-producing beta cells of the
pancreas. As a consequence, diabetic patients experience profound
metabolic derangements (hyperglycemia, ketosis and hyperlipemia) and
develop vascular and neurologic chronic complications[27-29].
These alterations can be only partially controlled by the exogenous
administration of insulin, which brings patients suffering and
inconvenience[30]. In recent years, engineering
insulin-secreting cell lines has received a great deal of interest
and some exciting advances have been made. But the application of
gene therapy to diabetes presents formidable challenges, one of
which is how the level of insulin is restricted within a very narrow
limit. Otherwise, if the engineered artificial β-cells were
transplanted into the body, the excessive insulin for metabolism
would cause hypoglycaemia[8], and even death. Several
inducible promoters originating from eukaryotic genes have been used
to deliver gene in a regulated manner. The substances inducing the
promoters include steroid hormones, oxygen, heavy metals, or
physical stimulus (such as radiation)[31, 32]. However,
most of these promoters are not suitable for clinical application
for various reasons: first, because these promoters are of mammalian
origin, the exogenous regulation of transgene through such a
promoter could at the same time affect the transcription of the host
endogenous genes; and second, the inducers of these promoters are
generally endogenous molecules (hormones, oxygen, etc.), the levels
of which cannot be modulated significantly and safely. Other
limitations include the potential toxicity or side effects of the
inducer. Therefore, an inducer/promoter system, which regulates the
transcription only of the transgene, without affecting endogenous
genes, is acceptable for gene therapy in humans.
The Tet-on system, which utilizes an E. coli gene
regulatory system[20], appears to fulfil the criteria for
clinical application for several advantages[33-40]:
(1)gene expression is easily regulated by administration of Dox;
(2)Dox is minimally toxic; and (3)Dox acts specifically on the
target gene, and does not activate other cellular genes. This
expression system has two critical components, the regulatory
plasmid and the response plasmid. The rtTA (reverse
tetracycline-controlled transactivator) expressed by the regulated
plasmid binds the TRE (tet-response element) in the response plasmid
and activates the transcription of the target gene in the presence
of Dox. Efrat et al [41] used the Tet-operon
regulatory system to generate a β-cell line in transgenic mice,
but there has been no report on the study of engineering non-β-cells
for the treatment of diabetes in this regard. Efrat constructed a
fusion protein, TETR-VP16 containing the tet repressor and the
activating domain of the herpes simplex virus protein VP16,which
converts the repressor into a transcriptional activator under the
control of the insulin promoter. Then, the transgenic mice of the
fusion protein were generated, whose β-cells could express the
TETR-VP16 protein conditionally. In a separate lineage of transgenic
mice, the simian virus 40 (SV40) large tumor (T) antigen (TAg) gene
was introduced under the control of the tet operator sequences and a
minimal promoter, which by itself is not sufficient for the
expression of Tag gene. Mice from the two lineages were then crossed
to generate double-transgenic mice. Expression of the TETR-VP16
protein in β-cells activated Tag transcription, resulting in
the development of β-cells tumors. A stable β-cell line
deriving from the tumor had the following characteristics: cell
incubated in the absence of Tet proliferated normally; and cell
incubation in the presence of Tet led to inhibition of
proliferation. Thus, it is feasible that the expression of insulin
gene can be regulated under the control of the proliferation of
these β-cells. In the present study, utilizing the Tet-on
system, we have established a cell line tet-293/Ins6 in which
insulin gene can be expressed conditionally by Dox treatment. Ten
μg·L-1 Dox can activate the tet-response element
and significantly enhance the expression of insulin gene in the
cells in a dose-dependent manner. Additionally, the RT-PCR results
showed that it is at 15min that 1mg·L-1 Dox could
activate the transcription of insulin gene in the tet-293/Ins6 cells
following the addition of Dox, and that the amount of insulin in the
media did not increase until 5 hours after the treatment of Dox,
suggesting that there is a periodic interval of time from the
process of gene transcription, translation, and protein synthesis,
processing to the final step, secreting the protein from the cells.
The strategy used here, by which a stable cell line with low
background and high Dox-induced expression of insulin was generated,
is more efficient and rapid for regulating insulin gene expression
as compared with Efrats' method[41], and will contribute
not only to the development of artificial β-cells for the
treatment of diabetes but to the generation of other
condition-secreting cell lines for gene therapy of human diseases.
However, responding to the variation of blood glucose
concentration, the expression level of insulin gene should be
regulated strictly for gene therapy of diabetes[42-44].
The tet-293/Ins6 cells still have their imperfections for replacing
islet transplantation in humans for the treatment of diabetes. Under
normal conditions, increasing glucose concentration in blood
stimulates β-cells to secrete insulin immediately; but the
tet-293/Ins6 cells release insulin by control of Dox at the
transcriptional level, the kinetics of feedback loops on
transcriptional changes is much slower than that of the secretory
response in β-cells. Therefore, based on the experiments we
have carried out, we are making efforts to establish an artificial
β-cell line in response to glucose stimulation with gene
engineering.
REFERENCES
1 Newgard CB, Clark S, BeltrandelRio H, Hohmeier
HE, Quaade C, Normington K. Engineered cell lines for insulin
replacement
in diabetes: current status and future prospects. Diabetologia
1997;40:S42-S47
2 Yuan Z, Wu GY, He YS, Shao CM, Zhan Y. Islet
separation and islet cell culture in vitro from human embryo
pancreas.
World J Gastroenterol 1999;5:458-460
3 Chen CQ, Lan P, Zhan WH. Studay on Fas-FasL
system in langerhans islets transplantation. Shijie Huaren Xiaohua
Zazhi
1999;7:422-423
4 Xu JT, Yan M, Yao YW, Wu R. Effect of
pentagastrin on IL-1β inducede inhibition of insulin secretion
in neonatal rat islets
Langerhans. World J Gastroenterol 2000;6(suppl 3):15
5 Regazzi R, Verchere CB, Halban PA, Polonsky KS.
Insulin production: from gene to granule. Diabetologial 1997;40:
B33-B38
6 Ferber S, Heimberg H, Brownlee M, Colton C.
Surrogate beta cells. Diabetologia 1997;40:B39-B43
7 Motoyoshi S, Shirotani T, Araki, E, Sakai K,
Kaneko K, Motoshima H, Yoshizato K, Shirakami A, Kishikawa H,
Shichiri M.
Cellular characterization of pituitary adenoma cell line
(AtT20 cell) transfected with insulin, glucose transporter
type 2 (GLUT2) and glucokinase genes: Insulin secretion in
response to physiological concentrations of glucose.
Diabetologia 1998;41:1492-1501
8 Falqui L, Martinenghi S, Berra C, Monti L, Leone
BE, Pozza G, Bordignnon C. Human proinsulin production in primary
rate
hepatocytes after retroviral vector gene transfer. J Mol Med
1999;77:250-253
9 Cheung AT, Dayanandan B, Lewis JT, Korbutt GS,
Rajotte RV, Bryer-Ash M, Boylan MO, Michael Wolfe M, Kieffer
TJ. Glucose-dependent insulin release from genetically
engineered K cells. Science 2000;290:1959-1962
10 Groskreutz DJ, Sliwkowski MX, Gorman CM. Genetically
engineered proinsulin constitutively processed and secreted as
mature,
Active insulin. J Biol Chem 1994;269:6241-6245
11 Gros L, Montoliu L, Riu E, Lebrigand L, Bosch F. Regulation
production of mature insulin by non-β-cells. Human Gene
Ther 1997;8:2249-2259
12 Falqui L, Martinenghi S, Severini GM, Corbella P, Taglietti
MV, Arcelloni C, Sarugeri E, Monti LD, Paroni R, Dozio N,
Pozza
G, Bordignon C. Reversal of diabetes in mice by implantation of
human fibroblasts genetically engineered
to release mature human insulin. Human Gene Ther
1999;10:1753-1762
13 Yamasaki K, Sakashi T, Nemoto M, Eto Y, Tajima N.
Differentiation-induced insulin secretion from nonendocrine cells
with engineered
human proinsulin cDNA. Biochem Bioph Res Co 1999;265:361-365
14 Dochert K. Gene therapy for diabetes mellitus. Clin Sci
1997;92:321-330
15 Efrat S. Prospects for gene therapy of insulin-dependent
diabetes mellitus. Diabetologia 1998;41:1401-1409
16 Rivera VM, Wang X, Wardwell S, Courage NL, Volchuk A,
Keenan T, Holt DA, Gilman M, Orci L, Cerasoli Jr F, Rothman
JE,
Clackson T. Regulation of protein secretion through controlled
aggregation in the endoplasmic reticulum.
Science 2000;287:826-830
17 Ye X, Rovera VM, Zoltick P, Cerasoli Jr F, Schnell MA, Gao
GP, Hughes JV, Gilman M, Wilson JM. Regulated delivery of
therapeutic protein after in vivo somatic cell gene transfer.
Science 1999;283:88-91
18 Lee HC, Kim SJ, Kim KS, Shin NC, Yoon JW. Remission in
models of type 1 diabetes by gene therapy using a single-chain
insulin analogue. Nature 2000;408:483-488
19 Grossen M, Butard H. Tight control of gene expression in
mammalian cells by tetracycline-responsive promoters. Proc
Natl
Acad Sci USA 1992;89:5547-5551
20 Song KC, Haller GW, Giauffret D, Germana S, Reeves SA, Levy
J, Sachs D, Leguern C. Regulated expression of an MHC
class
II gene from a promoter-inducible retrovirus. Human Gene Ther
2000;11:1961-1969
21 Dressel R, Lubbers M, Walter L, Herr W, Gunther E. Enhanced
susceptibility to cytotoxic T lymphocytes without increase
of
MHC class Ⅰ
antigen expression after conditional overexpression of heat shock
protein 70 in target cells.
Eur J Immunol 1999;29:3925-3935
22 Hagihara Y, Saitoh Y, Arita N, Eguchi Y, Tsujimoto Y,
Yoshimine T, Hayakawa T. Long-term functional assessment of
encapsulated cells transfected with tet-on system. Cell
Transplant 1999;8:431-434
23 Krauthauser CM, Hall LM, Wexler RS, Slee AM, Mitra J,
Enders GH, Kerr JS. Regulation of gene expression and cell
growth
in vivo by tetracycline using the hollow fiber assay.
Anticancer Res 2001;21:869-872
24 Lottmann H, Vanselow J, Hessabi B, Walther R. The tet-on
system in transgenic mice: inhibition of the mouse pdx-1
gene
activity by antisense RNA expression in pancreatic -cells. J Mol Med
2001;79:321-328
25 Milo-Landesman D, Surana M, Berkovich I, Compagni A,
Christofori G, Fleischer N, Efrat S. Correction of hyperglycemia
in
diabetic mice transplanted with reversibly immortalized pancreatic
cells controlled by the tet-on regulatory system.
Cell
Transplant 2001;10:645-650
26 Lin L, Lu XZ, Zhao ZQ. Electrogastrography in patients with
disordered gastric motility in diabetes and effect of cisapride.
China Natl J New Gastroenterol 1996;2(suppl 1):71-72
27 Wang WM, Lu GX.Research on the treatment of diabetic
gastroparesis by erythromycin. China Natl J New Gastroenterol
1996;2(suppl 1):130
28 Quigley EMM. The evaluation of gastrointestinal function in
diabetic patients. World J Gastroenterol 1999;5:277-282
29 Gu YP, Gu JY, Li JS. Pancreaticoduodenal transplantation
with portal venous and enteric drainage in rats. World J
Gastroenterol 2000;6:914-916
30 Sartoh Y, Eguchi Y, Hagihara Y, Arita N, Watahiki M,
Tsujimoto Y, Hayakawa T. Dose-dependent doxycycline-
mediared adrenocorticotropic hormone secretion from
encapsulated Tet-on proopiomelanocortin Neuro2A cells in the
subarachnoid space. Human Gene Ther 1998; 9:997-1002
31 Paillard F. “Tet-on”: a gene switch for the exogenous
regulation of transgene expression. Human Gene Ther
1998; 9:983-985
32 Bachl J, Carlson C, Gray-Schopfer V, Dessing M, Olsson C.
Increased transcription levels induce higher mutation rates
in
a hypermuting cell line. J Immunol 2001;166:5051-5057
33 Molin M, Shoshan MC, hman-Forslund K, Linder S, Akusj rvi
G. Two novel adenovirus vector systems permitting
regulated
protein expression in gene transfer experiments. J Virol
1998;72:8358-8361
34 Kashima Y, Miki T, Minami K, Seino S. Establishment of a
tet-on gene expression system in glucose-responsive and
-unresponsive MIN6 cells. Diabetes 2001;50:S133
35 Levy LM, Warr O, Attwell D. Stoichiometry of the glial
glutamate transporter GLT-1 expressed inducibly in a Chinese
Hamster
ovary cell line selected for low endogenous Na+-dependent glutamate
uptake. J Neurosci 1998;18:9620-9628
36 Zhu HJ, Iaria J, Sizeland AM. Smad7 differentially
regulates transforming growth factor β-mediated signaling
pathway.
J Biol Chem 1999;274:32258-32264
37 Wang DY, Grammer R, Cobbs CS, Stewart JE Jr, Liu ZY, Rhoden
R, Hecker TP, Ding Q, Gladson CL. P125 focal adhesion
kinase promotes malignant astrocytoma cell proliferation
in vivo. J Cell Sci 2000;113:4221-4230
38 Kobayashi T, Sawa H, Morikawa J, Zhang W, Shiku H. Bax
inducetion activates apoptotic cascade via mitochondrial
cytochrome crelease and bax overexpression enhances apoptosis
induced by chemotherapeutic agents in DLD-1 colon
cancer cells. Jpn J Cancer Res 2000;91:1264-1268
39 Serguera C, Bohl D, Rolland E, Provost P, Heard JM. Control
of erythropoietin secretion by doxycycline or mifepristone in
mice
bearing polymer-encapsulated engineered cells. Human Gene Ther
1999;10:375-383
40 Serguera C, Bohl D, Rolland E, Provost P, Heard JM. Control
of erythropoietin secretion by doxycycline or mifepristone in
mice
bearing polymer-encapsulated engineered cells. Human Gene Ther
1999;10:375-383
41 Efrat S, Fusco-DeMane D, Lemberg H, Emran OA, Wang XR.
Conditional transformation of a pancreatic β-cell line
derived
from transgenic mice expressing a tetracycline-regulated oncogene.
Proc Natl Acad Sci USA 1995;92:3576-3580
42 Mitanchez D,Doiron B,Chen R,Kahn A.Glucose-stimulated genes
and prospects of gene therapy for typeⅠdiabetes.
Endocr Rev
1997;18:520-540
43 Tiedge M, Elsner M, Mcclenaghan NH, Hedrich HJ, Grube D,
Klempnauer J, Lenzen S. Engineering of a glucose-responsive
surrogate cell for insulin replacement therapy of
experimental insulin-dependent diabetes. Human Gene Ther
2000;11:403-414
44 Barry S, Ramesh N, Lejnieks DV, Simonson W, Kemper L,
Lernmark A, Osborne WRA. Glucose-regulated insulin
expression
in diabetic
rats. Human Gene Ther 2001;12:131-139
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