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Molecular Medicine and Gene Therapy Unit, University of Manchester School of Medicine (J.R.S.-A., J.C.W., D.S., P.R.L., M.G.C.), Manchester, United Kingdom M13 9PT; and AstraZeneca (J.S.), Macclesfield, Cheshire, United Kingdom SK10 4TG
Address all correspondence and requests for reprints to: Prof. M. G. Castro or Prof. P. R. Lowenstein, Molecular Medicine and Gene Therapy Unit, Room 1.302, Stepford Building, University of Manchester School of Medicine, Oxford Road, Manchester, United Kingdom M13 9PT. E-mail: mcastro@fs1.scg.man.ac.uk or lowenstein{at}man.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, in situ transduction of the AP gland has been shown using recombinant adenoviral vectors (RAds) after transauricular (5) or transcranial stereotaxic delivery (6, 7, 8). No cytotoxicity or loss of AP hormonal functions is apparent after vector delivery (7), indicating that RAds would be a useful tool for studying gene function within the AP gland and could be further developed for gene therapy applications.
Targeted transgene expression to predetermined cell types after adenovirus-mediated delivery to the AP gland could be achieved through two alternative methods. The retargeting of RAds so that they bind to a specific membrane receptor can be achieved, and this would enable viral targeting to a given cell population (9). An alternative to direct viral targeting is the use of cell type-specific promoters that limit transgene expression to a particular cell type. RAds driving transgene expression from cell type-specific promoters have been successfully used (6, 7, 8, 10, 11, 12).
To generate a system in which transgene expression can be regulated, we used an inducible transcription system, i.e. the tetracycline-dependent regulatory (tet) system (13). Gene expression using variations of this system can be switched on or off by the presence of tetracycline, enabling controlled transgene expression during any given time period. In the tet off system a synthetic tetracycline- dependent trans-activator (tTA) is able to bind to and activate transcription from a tetracycline-responsive element (TRE) promoter in the absence, but not the presence, of tetracycline. In the tet on system a mutated tetracycline-dependent transactivator (rtTA) is able to bind to and activate transcription from the TRE promoter in the presence, but not the absence, of tetracycline. Although when used in transgenic mouse lines regulation from the tet system can be variable (14), its use within RAds has provided successful regulation of transgene expression (15, 16, 17, 18).
To test whether both cell type-specific and regulatable transient transgene expression could be attained in the AP gland in vivo, a dual adenoviral vector system was developed. The lactotroph-specific human PRL (hPRL) promoter (bp +14 to -4429) drove the expression of a nuclear localized tTA [tTA(nls)] from one RAd, and the TRE promoter drove expression of the ß-galactosidase reporter from another RAd. The cell type specificity and regulatable transgene expression of this system were assessed in cell lines, primary AP cultures, and the AP gland in situ.
Our data demonstrate that the hPRL promoter can be combined with the tet off system within RAds to produce combined cell type-specific and regulatable transgene expression in lactotroph tumor cells, lactotroph cells within primary AP cultures, and in vivo, within the AP gland in situ. This system should provide an alternative to knockout or transgenic animals for studies of molecular and biochemical mechanisms within the AP gland. It should also prove useful for the development of therapeutic treatments within the AP gland, such as the treatment of hypopituitarism, that rely upon the controlled pulsatile release of AP hormones.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of recombinant adenoviruses
Previously the full-length rat PRL promoter containing proximal
(+33 to -422 bp) and distal (-1500 to -1800 bp) positive regulatory
elements with 90% homology to the hPRL promoter (21) was
shown to restrict expression of nerve growth factor, transforming
growth factor-
, or hGH to lactotrophic (22, 23) and
mammosommatotrophic (24) cells in transgenic mice. As it
was shown that the distal and proximal elements of the rat promoter
were essential for strong lactotroph-specific expression, we decided to
use a 4.4-kb hPRL promoter fragment (+14 to -4429 bp) that contains
the proximal (-40 to -250 bp), the distal (-1300 to -1750 bp), and
a portion of the super distal (-3500 to -5000 bp) elements of the
hPRL promoter (21). The 4.4-kb hPRL fragment of the hPRL
promoter, the tTA(nls) open reading frame (15) from
pCANtTA (Dr. H. Hamada, Department of Molecular Biotherapy Research,
Cancer Institute, Tokyo, Japan), and the simian virus 40
polyadenylation signal were cloned as an XbaI fragment into
p
E1sp1A (Microbix Biosystems, Inc.) to create the
pE1-hPrl-tTA(nls) shuttle vector. The CAG-tTA(nls) expression
cassette from the plasmid pCANtTA, containing the CAG (chicken
ß-actin/hCMV) promoter, was inserted as a
ClaI/BglII fragment into pE1sp1A to create the
pE1-CAG-tTA(nls) shuttle vector. The TRE-ß-galactosidase cassette
from pTRE-ß-galactosidase (CLONTECH Laboratories, Inc.,
Palo Alto, CA) was inserted as an XhoI/HindIII
fragment into pE1sp1A to create the pE1-TRE-ß-galactosidase
shuttle vector. RAd-CAG-tTA(nls), RAd-hPRL-tTA(nls), and RAd-TRE-ß-
galactosidase were generated by homologous recombination in 293
cells of the respective shuttle vector with either pJM17 or pBHG10
(Microbix Biosystems, Inc.). Characterization, production of high titer
stocks, purification by double cesium chloride density gradient
separation, and titration of viruses were carried out as previously
described (25, 26). Stocks were found to be free of
replication competent adenovirus using a supernatant rescue assay able
to detect 1 replication competent virus within
109 recombinant viruses (27).
Adenovirus preparations were ascertained to be endotoxin
(lipopolysaccharide) free, according to the criteria of Cotten et
al. (28), using the E-TOXATE assay
(Sigma-Aldrich Corp., Dorset, UK).
Quantitative in vitro analysis of ß-galactosidase activity
Cells were plated in 48-well plates at a density of 1 x
104 cells/well 1 day before infection in the
presence or absence of the tetracycline analog doxycycline (500 ng/ml).
Primary rat AP cultures were incubated for 7 days before infection, and
doxycycline (500 ng/ml) was administered 24 h before infection.
Cell lines were infected with virus combinations at ratios of 10:1 and
1:1, with appropriate multiplicities of infection (MOIs) of 500:50 or
50:50, whereas primary rat AP cultures were infected with virus
combinations at a ratio of 10:1 with a respective MOI of 500:50. Cells
and primary rat AP cultures were infected with virus at constant MOIs
for each experiment and at levels that do not cause cytotoxicity, as
determined previously (data not shown). On the day of infection cells
were counted and infected at appropriate MOI values to produce ratios
of tTA(nls):TRE of 10:1 [MOI of 500 (5 x
106 infectious units (iu) to MOI of 50
(5 x 105) of each vector] or a ratio of
ETA (nls):TRE of 1:1 [MOI of 50 (5 x 105
iu) to MOI of 50 (5 x 105) of each
vector], with virus combinations of
RAd-CAG-tTA(nls):RAd-TRE-ß-galactosidase or
RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase. Infected cells were left
for 48 h after infection, after which cell lysates were assessed
for relative ß-galactosidase activity as described previously
(6, 26). n = 4 for each group, and each
experiment was repeated at least twice.
Delivery of RAds to the anterior pituitary gland in vivo
Male 8-week-old Buffalo rats were house-bred at the University
of Manchester Biological Safety Unit. All animals had free access to
food and water and were kept under a 12-h light, 12-h dark cycle with
constant housing temperature and humidity. Experiments were conducted
according to the United Kingdom Animal (Scientific Procedures) Act of
1986. One day before surgery rats were given water containing 1%
sucrose in the absence or presence of doxycycline (2 mg/ml). The
procedure for stereotaxic delivery of RAds to the AP gland in
vivo has been described in detail previously (4).
Briefly male 8-week-old Buffalo rats were anesthetized with halothane,
placed in a stereotaxic frame, and injected six times in the AP gland
(three sites per lobe) with a total dose of 1 x
108 iu virus. The specified dose was chosen
because it has previously been shown to produce high levels of gene
transfer in the AP gland without causing cytotoxicity (7).
A volume of 1 µl was injected per site over a period of 1 min.
Animals were then given 10 ml saline, sc, and allowed to recover. After
3 days animals were given a lethal injection of pentobarbitone and
perfused transcardially with Tyrode’s solution (132
mM NaCl, 1.9 mM
CaCl2, 0.32 mM
NaH2PO4, 5.56
mM glucose, 11.6 mM
NaHCO3, and 2.68 mM KCl),
and pituitary glands were removed and postfixed by placing them in 4%
paraformaldehyde solution. Pituitaries were then paraffin-embedded, cut
into 5-µm sections using a microtome, and placed onto
3-aminopropyltriethoxysilane-coated slides.
In the experiments in which expression of ß-galactosidase was switched on and off, male 8-week-old Buffalo rats were given water containing 1% sucrose in the absence or presence of doxycycline (2 mg/ml) 1 day before surgery. Four days after surgery animals initially given water with doxycycline were given water without doxycycline, and animals that had initially been given water without doxycycline were given water with doxycycline. Groups of animals were then killed 11 and 18 days after surgery.
Fluorescence immunohistochemical detection of ß-galactosidase
and hormones
Protocols for fluorescent immunohistochemical detection within
primary pituitary cultures and paraffin-embedded pituitary sections
have been described in detail previously (5, 6, 20). The
primary antibody for detecting ß-galactosidase was a polyclonal
rabbit anti-ß-galactosidase (1:500) provided by Dr. R. Goya
(University of La Plata School of Medicine, La Plata, Argentina). The
primary antibodies to hormones were polyclonal: guinea pig antirat PRL
(1:100), guinea pig antihuman GH (1:100), and guinea pig antirat -LH
(1:100) provided by Dr. A. F. Parlow and the NIDDK National
Hormone and Pituitary Program (Bethesda, MD). Secondary antibodies were
goat antiguinea pig fluorescein isothiocyanate (1:100) and goat
antirabbit tetramethyl rhodamine isothiocyanate (1:100) from
Jackson ImmunoResearch Laboratories, Inc. (West Grove,
PA). Fluorescence images were taken using an Olympus Corp.
Vanox-T microscope (New Hyde Park, NY) with BP490:O515 and BP545:O590
exciter:barrier filter combinations for fluorescein isothiocyanate and
tetramethyl rhodamine isothiocyanate, respectively.
Statistical analysis
In vitro experimental results were analyzed using
Student’s two-tailed t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). RAds
driving expression of a nuclear localized transactivator from the
ubiquitous chicken ß-actin/human cytomegalovirus (hCMV) fusion
(CAG) promoter [RAd-CAG-tTA(nls)] or the lactotroph-specific hPRL
promoter [RAd-hPRL-tTA(nls)] were generated (Fig. 1, A and B). A RAd
with the TRE driving expression of the ß-galactosidase reporter gene
(RAd-TRE-ß-galactosidase) was also generated (Fig. 1C). The presence
of the expression cassettes within each RAd was confirmed by Southern
blotting HindIII-digested viral DNA using specific probes
for each expression cassette. Bands of the expected size from each
viral DNA digest were positive for the respective expression cassettes
(Fig. 1, A–C).
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). It must also be noted that the
two independent infectious events needed per cell from our dual
adenovirus system coupled to the fact that AtT20s are not very
receptive to adenovirus infection mean that AtT20 cells show
comparatively low levels of ß- galactosidase expression. Similar
results were obtained when using a single adenovirus vector expressing
ß-galactosidase (6). When BHK, AtT20, CNS-1, and
GH3 cells were infected with
RAd-CAG-tTA(nls):RAd-TRE-ß-galactosidase at a ratio of 10:1 in the
absence of doxycycline, specific ß-galactosidase activity was induced
to 103 ± 8.1, 0.017 ± 0.001, 94.8 ± 2.2, and
40.4 ± 2.6 U/µg protein, respectively (Fig. 2). When BHK,
CNS-1, and AtT20 cells were infected with
RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase at ratios of 1:1 or 10:1 in
the absence of doxycycline, no induction of specific ß-galactosidase
activity was seen (Fig. 2). When GH3 cells were
infected with RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase at a ratio of
1:1 in the absence of doxycycline, specific ß-galactosidase activity
was induced to 1.7 ± 0.06 U/µg protein (Fig. 2). When
GH3 cells were infected with
RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase at a ratio of 10:1 in the
absence of doxycycline, specific ß-galactosidase activity was induced
to 6.4 ± 0.1 U/µg protein (Fig. 2). Negligible basal levels of
specific ß-galactosidase activity were seen in all groups in the
presence of doxycycline.
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and Table 1), FSH, TSH, or ACTH (data not shown) in
the absence, but not in the presence, of doxycycline. Infection with
RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase resulted in expression of
ß-galactosidase in fewer cells, in the absence, but not the presence,
of doxycycline. Expression was restricted mainly to lactotrophic (PRL-
expressing) cells and a subpopulation of cells expressing GH that
are likely to be mammosomatotrophs and also express PRL (Fig. 3B and
Table 1). Of the total ß-galactosidase- positive cell population,
about 87% were lactotrophs and 13% were mammosomatotrophs. This cell
type specificity achieved with
RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase explains the lower number
of cells expressing ß-galactosidase compared with
RAd-CAG-tTA(nls):RAd-TRE-ß- galactosidase and is expected,
because PRL-positive cells typically make up approximately 35% of
primary anterior pituitary cultures. No ß-galactosidase expression
was seen in cells expressing the other AP hormones, i.e. LH
(Fig. 3B) or FSH, TSH, or ACTH (data not shown), in either the presence
or absence of doxycycline.
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). When cultures were infected with
RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase in the absence of
doxycycline, specific ß-galactosidase activity was induced to
1.03 ± 0.08 U/µg protein (Fig. 4). Negligible basal levels of
specific ß-galactosidase activity were seen in all groups in the
presence of doxycycline. The
RAd-CAG-tTA(nls):RAd-TRE-ß-galactosidase-infected cultures showed a
21-fold increase in induction of ß-galactosidase activity compared
with RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase-infected cultures
(Fig. 4). Higher levels of ß-galactosidase expression obtained in the
on state with RAd-CAG-tTA(nls):RAd-TRE-ß-galactosidase compared
with RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase were expected, because
expression from the hPRL promoter is only seen in lactotrophic cells,
which represent about 35% of the total endocrine cell population
within primary AP cultures (Table 1
), although the lower percentage of
cells in which the transgene is expressed cannot explain the 21-fold
difference observed.
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).
Infection with RAd-CAG-tTA(nls):RAd-TRE-ß-galactosidase resulted in
expression of ß-galactosidase in cells expressing PRL, GH, or LH
(Fig. 5B) or FSH, TSH, and ACTH (data not shown) in the absence, but
not the presence, of doxycycline. Infection with
RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase resulted in expression of
ß-galactosidase in fewer cells in the absence, but not the presence,
of doxycycline. Expression was restricted mainly to lactotrophic cells
and a subpopulation of cells expressing GH that are likely to be
mammosomatotrophs and also express PRL (Fig. 5C and Table 2). The cell type specificity
displayed by the RAd-hPRL-tTA(nls):RAd-TRE-ß-galactosidase dual virus
system explains the lower number of cells expressing ß-galactosidase
compared with RAd-CAG-tTA(nls):RAd-TRE-ß-galactosidase and is
expected, as PRL-producing cells comprise approximately 32% of all
endocrine AP cell types around the injection sites of these animals
(Table 2). As ß-galactosidase expression was seen in 25 ± 2.6%
of PRL-positive cells (Table 2), then approximately 6% of the total
cell population were positive for both PRL and ß-galactosidase. No
ß-galactosidase expression was seen in cells expressing the other AP
hormones, i.e. LH (Fig. 5C and Table 2) or FSH, TSH, or ACTH
(data not shown) in either the presence or absence of
doxycycline.
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). The expression
of ß-galactosidase was then assessed 11 and 18 days after surgery. In
the animals that received doxycycline 4 days after surgery,
ß-galactosidase expression was found in a very small number of cells
sparsely scattered around the AP gland at 11 days, although almost all
of the ß-galactosidase expression was switched off (Fig. 6). Eighteen
days after surgery, no ß-galactosidase-positive cells were found in
the presence of doxycycline (Fig. 6). In the animals that had
doxycycline withdrawn at 4 days, expression of ß-galactosidase was
seen at both 11 and 18 days (Fig. 6). The levels of ß-galactosidase
expression seen in the absence of doxycycline on days 11 and 18 showed
a gradual decline over time compared with the levels on day 3 (Fig. 5A), in accordance with our previous observations (7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although both cell type-specific promoters and the tetracycline-responsive system have been successfully used to express transgenes within RAds (6, 7, 8, 17, 18, 29), this is the first report on using a combined cell type-specific and regulated system within the AP gland in vitro and in vivo. We inserted the hPRL-tTA(nls) and TRE-ß-galactosidase cassettes in two separate RAds so that we could address the issues of optimal tTA(nls) to TRE ratio and promoter interference.
There are conflicting data indicating the ratio of
trans-activator to TRE at which the best regulation of the
tet system is seen. Previously, it had been shown that an excess of TRE
is needed to achieve efficient regulation from RAds when using either
the tTA or rtTA trans-activator (17). Corti
et al. (18), however, showed that a 1:1 ratio
of tTA to TRE is able to mediate tight control of gene expression,
whereas previous results from our laboratory have consistently shown
that an excess of tTA to TRE in a two plasmid or adenoviral system
improves regulated gene expression compared with other ratios
(30) (data not shown). Unlike the studies by Harding
et al. (17, 31) and Corti et al.
(18), the tTA used in our system contains a nuclear
localization signal, and as the compartmental concentrations of tTA or
TRE within the cell are likely to influence the control and levels of
tet-regulated expression, this may play an important role in both the
regulation and the leakiness obtained with this system. Previously, the
addition of a nuclear localization signal to the rtTA resulted in
reduced induction and increased basal gene expression from RAds
(31); however, the addition of an nls to the tTA caused
increased induction and decreased basal gene expression from RAds at a
variety of tTA to TRE ratios (15). It is possible that an
excess of nuclear rtTA may result in a reduced level of TRE binding,
whereas excess nuclear tTA may result in increased TRE binding. The
exact reasons behind this anomaly are unclear, so with these factors in
mind we decided to determine the tTA(nls) to TRE ratio that would
provide the highest levels of transgene expression without causing
leaky gene expression. When tTA(nls) to TRE ratios of 10:1 and 1:1 were
used in pituitary tumor cell lines we saw tight regulation with both
ratios, but higher induction levels with a ratio of 10:1, and therefore
decided to use this ratio for the subsequent experiments. It is
possible that the increased induction levels of gene expression seen at
a ratio of 10:1 are caused by increased intracellular levels of
tTA(nls) up until a point at which TRE induction plateaus and/or
becomes inhibited. Such a scenario might explain the lack of increase
in induction between the 1:1 and 10:1 ratios when using the stronger
CAG promoter in GH3 cells (Fig. 2), as this ratio
in GH3 cells, and not BHK, AtT20, or CNS-1 cells,
may produce inhibitory levels of tTA(nls). Alternatively, the levels of
tTA(nls) reached within GH3 cells using the CAG
promoter at a 10:1 ratio may be toxic.
In addition to its use in assessing the optimal tTA to TRE ratio, our system was designed as a dual adenovirus system to circumvent problems of interference with the transcriptional or regulatory elements. It has been suggested that by placing the promoter driving expression of the tTA in tandem with or in the same backbone as the TRE promoter, nonspecific induction of gene expression can occur (32), and this may be coupled to reduced inducibility. Although leaky expression is not observed when an alternative regulatory system to the tet system is used in tandem within a RAd (33), this may not be the case with the tet system, as tail to tail insertion of the tTA/TRE cassettes within a RAd (34) resulted in leaky gene expression. The insertion of an insulator sequence between the two cassettes (18) or the division of the two cassettes between two RAds (15, 17, 31) appears to avoid this interference of the regulatory elements within a single RAd, and our results confirm the validity of the later approach. It has also been suggested that the backbone of all RAd vectors may be deleterious to either transcriptional or regulatory systems due to the presence of the E1A enhancer within the lefthand ITR. As ß-galactosidase expression from our dual RAd system was both cell type specific and tightly regulated, we concluded that the transcriptional and regulatory functions of our dual virus system were not affected by the RAd genetic backbone. It is likely that by placing the tTA(nls) and TRE elements on two different RAds we have avoided the basal promoter leakage and weak repression of expression previously seen with the tet system within a single herpes virus amplicon vector (35).
Although ß-galactosidase expression was seen in GH- producing AP cells using our system, this is probably due to the presence of mammosomatotrophic cells within the AP gland that secrete both GH and PRL (36) and not to interference of the hPRL promoter elements. Previously, transgenic mice expressing hGH from a full-length rat PRL promoter restricted expression of hGH to lactotrophs and mammosomatotrophs (24). Although it cannot be excluded that this expression may be due to hPRL expression in somatotrophs, it would seem unlikely, as the number of ß-galactosidase/GH-positive cells is considerably lower than the number of ß- galactosidase/Pro cells, and male rats generally have higher numbers of somatotrophs than lactotrophs. Also somatotrophs are readily infected by adenoviruses (5, 6, 7, 8, 12, 20) and when the ubiquitous CAG promoter was used similar levels of transduction (60–65%) were seen in all cell types, so a lack of somatotroph infectivity cannot be the reason for the reduced levels of ß-galactosidase expression seen in somatotrophs compared with lactotrophs when using the hPRL promoter, if this were the case. Furthermore, it has been shown previously that mammosomatotrophs in mice and rats make up between 1–30% of the total GH-positive population in the AP gland (22, 37, 38). Both in vitro (13%) and in vivo (5%) using the hPRL promoter, we observed ß- galactosidase expression in a proportion of the total GH-positive cells that correlates with this observation.
For such a cell type-specific system to be of use in a therapeutic setting, the levels of expression would have to be high enough to achieve a beneficial therapeutic outcome, so the efficiency of the hPRL promoter was assessed quantitatively. Although transgene expression from the hPRL promoter within this study appears to be restricted to PRL-secreting cells, it is apparent that the relative levels of inducible expression seen in vitro are lower than those obtained with the ubiquitous CAG promoter. Although a lower number of ß-galactosidase-transduced cells would be expected using the hPRL promoter in mixed cell populations due to the cell type-specific nature of the promoter, this does not explain the lower levels of specific ß-galactosidase activity, compared with the CAG promoter, seen at both ratios in PRL-secreting GH3 cells in the absence of doxycycline. Additionally the 21-fold increase in specific ß-galactosidase activity between hPRL and CAG in primary AP cultures in the absence of doxycycline does not correlate with the 2.4-fold increase (data not shown) in total endocrine cells in primary AP cultures expressing ß-galactosidase when using the CAG promoter. Even if ß-galactosidase expression from the CAG promoter is seen in 100% of the fibroblasts that account for the remaining 65% of cells within primary AP cultures at 7 days (data not shown), this would only result in an 8.6-fold increase (data not shown) in total cells expressing ß-galactosidase. It appears that in vitro the hPRL promoter is a weaker promoter than the CAG promoter, and this correlates with previous in vitro and in vivo observations (6, 7).
As our system is reliant upon a cell being infected by two different viruses, one of its main limitations is the level of cell type-specific transduction achievable (64% of PRL-positive cells in vitro and 25% in vivo). By increasing the amount of virus used it would be possible to transduce a higher number of cells, but there is a level, both in vitro and in vivo, at which adenovirus becomes toxic, and the levels of virus used in our experiments were deliberately chosen to be below those thresholds (as determined by us previously, data not shown) (7). Even so it would be difficult to achieve an overall transduction efficiency in vivo much greater than 25%, because the cells closer to the injection site are more accessible to infection and vice versa. Even so, levels of 25% expression would be sufficient for many therapies, such as the production of hormones, which are secreted molecules. In this case the tet system could be used to regulate hormone release to therapeutic levels independently of the number of cells in which the hormone is expressed. Also, certain tumor treatments, such as the suicide gene therapy approaches (i.e. herpes simplex virus 1-thymidine kinase), do not rely upon high transduction efficiencies, because they have efficient bystander-killing effects.
It has previously been shown using dual adenovirus tet off or on regulated systems that expression can be switched on and off in the brain. Harding et al. (17) showed that using the tet off system enhanced green fluorescent protein expression was reduced 5 days after doxycycline administration and abolished by 10 days, whereas using the tet on system, enhanced green fluorescent protein expression was reduced 7 days after doxycycline withdrawal and abolished by 10 days. With our system we also saw a slow complete switching off of transgene expression in the presence of doxycycline that took more than 7 days. Although this could be caused by the slow switching off of transcription, due to poor accessibility of doxycycline to the AP gland, it is more likely to be due to the half-life of ß-galactosidase, as expression of ß-galactosidase in the AP gland is prevented 4 days after a single doxycycline administration. Unlike when it is switched off, the switching on of transgene expression can be seen at 7 days, although the levels are reduced by 14 days. This quicker switching from one state to the other may be due to the shorter half-life of doxycycline within the body, and the reduced levels seen from 7–14 days are expected because we have previously observed a drop in RAd-mediated AP- directed transgene expression over time (7).
To aid the development of our understanding of AP function and to advance current gene therapy strategies for therapeutic treatment of pituitary diseases, advances in gene delivery technology need to be made. These ought to address putative side-effects (39, 40, 41), efficiency, and longevity of transgene expression. With this in mind, we have developed a combined cell type-specific and regulatable expression system that is able to efficiently express a transgene in a cell type-specific and regulatable fashion within the AP gland in situ. Although the combined use of cell type-specific and regulatable systems has been shown previously in transgenic animals in the brain (42, 43) and liver (44) or from RAds in the liver (33), tumor cells (45), or brain (30, 46), to our knowledge this is the first reported use of a combined lactotroph-specific and regulatable expression system. The data presented in this paper indicate that transcriptional and reversible inducible transgenesis can be achieved using adenoviral vectors that allow spatial and temporal restriction of transgene expression within the adult AP gland in vivo.
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Acknowledgments |
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Footnotes |
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2 J.R.S.-A., J.C.W., and D.S. contributed equally to this work and
should be considered first authors.
3 Present address: Children’s Hospital Research Foundation, W531
700 Children’s Drive, Columbus, Ohio 43205.
4 Present address: Cedars-Sinai Medical Center, Burns and Allen
Research Institute, Gene Therapeutics Institute, 8700 Beverly
Boulevard, Los Angeles, California 90048-0750.
5 Fellow of The Lister Institute of Preventive Medicine.
Received October 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene expression. Cancer Res 57:3339–3343 expression to
pituitary lactotrophs in transgenic mice results in selective
lactotroph proliferation and adenomas. Endocrinology 136:4479–4488[Abstract]
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 M. P. Gillam, M. E. Molitch, G. Lombardi, and A. Colao Advances in the Treatment of Prolactinomas Endocr. Rev., August 1, 2006; 27(5): 485 - 534. [Abstract] [Full Text] [PDF]
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M Candolfi, G Jaita, D Pisera, L Ferrari, C Barcia, C Liu, J Yu, G Liu, M G Castro, and A Seilicovich Adenoviral vectors encoding tumor necrosis factor-{alpha} and FasL induce apoptosis of normal and tumoral anterior pituitary cells. J. Endocrinol., June 1, 2006; 189(3): 681 - 690. [Abstract] [Full Text] [PDF]
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W. Xiong, S. Goverdhana, S. A. Sciascia, M. Candolfi, J. M. Zirger, C. Barcia, J. F. Curtin, G. D. King, G. Jaita, C. Liu, et al. Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses J. Virol., January 1, 2006; 80(1): 27 - 37. [Abstract] [Full Text] [PDF]
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E. J. Lee and J L. Jameson Gene therapy of pituitary diseases J. Endocrinol., June 1, 2005; 185(3): 353 - 362. [Abstract] [Full Text] [PDF]
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L. Barzon, M. Boscaro, and G. Palu Endocrine Aspects of Cancer Gene Therapy Endocr. Rev., February 1, 2004; 25(1): 1 - 44. [Abstract] [Full Text] [PDF]
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 M. S. Sarac, S. Windeatt, M. G. Castro, and I. Lindberg Intrapituitary Adenoviral Administration of 7B2 Can Extend Life Span and Reverse Endocrinological Deficiencies in 7B2 Null Mice Endocrinology, June 1, 2002; 143(6): 2314 - 2323. [Abstract] [Full Text] [PDF]
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) recombine to produce a
recombinant genome with an insertion in E1. A schematic representation
of each linear recombinant genome is shown below along
with the respective insert and the region used to make a probe for
Southern blotting. Each recombinant genome digested with
HindIII is also shown alongside the respective Southern
blot, indicating the presence of each expression cassette. Viral genome
bands positive for the respective expression cassettes are indicated
along with their respective sizes.
