The Tripartite Basal Enhancer of the Gonadotropin-Releasing Hormone (GnRH) Receptor Gene Promoter Regulates Cell-Specific Expression Through a Novel GnRH Receptor Activating Sequence
Dawn L. Duval,
Scott E. Nelson and
Colin M. Clay
Animal Reproduction and Biotechnology Laboratory Department of
Physiology College of Veterinary Medicine and Biomedical
Sciences Colorado State University Fort Collins, Colorado
80523
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ABSTRACT
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The molecular mechanisms regulating restricted
expression of GnRH receptor and gonadotropin subunit genes to
gonadotrope cells have been the focus of intense interest. Using
deletion and mutational analysis we have identified a tripartite
enhancer that regulates cell-specific expression of the GnRH receptor
gene in the gonadotrope-derived
T31 cell line. Individual
elements of this enhancer include binding sites for steroidogenic
factor-1; activator protein 1 (AP-1); and a novel element referred to
as the GnRH receptor activating sequence (GRAS). Mutation of each
element alone results in loss of approximately 60% of promoter
activity. Combinatorial mutations of any two elements decreases
promoter activity by approximately 80%. Finally, mutation of all three
elements reduces promoter activity to a level not different from
promoterless vector. Using 2-bp mutations, we have defined the
functional requirements for transcriptional activation by GRAS. The
core motif of GRAS is at -391 to -380 bp relative to the start site
of translation and has the sequence 5'-CTAGTCACAACA-3'. Three copies of
GRAS or GRAS with a 2-bp mutation (µGRAS) were cloned into a
luciferase expression vector immediately upstream of the thymidine
kinase minimal promoter (TK) and tested for expression in
T31
cells. When compared with TK promoter alone, activity of 3xGRAS-TKLUC
was increased by more than 5-fold while activity of 3xµGRAS-TKLUC was
unchanged. When 3xGRAS-TKLUC was transfected into a variety of
nongo-nadotrope cell lines, it did not increase activity of the TK
promoter. We propose that basal activity of the GnRH receptor gene is
regulated by a tripartite enhancer, and the key component of this
enhancer is an element, GRAS, that activates transcription in a
cell-specific fashion.
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INTRODUCTION
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The ordered emergence of corticotropes, thyrotropes, somatotropes,
mammotropes, and gonadotropes in the anterior pituitary gland and the
selective secretion of their respective hormones provide an excellent
opportunity to study molecular mechanisms underlying cell-specific gene
regulation (1). Definition of the molecular codes that direct
cell-specific gene expression of the common glycoprotein hormone
-subunit, the unique LH and FSH ß-subunits, and the GnRH receptor
(GnRH-R) to gonadotropes has advanced rapidly during the past few
years. Key to this progress has been use of transgenic technologies (2, 3) and availability of the gonadotrope-derived
T31 cell line that
expresses GnRH-Rs and the
-subunit, but not the specific LH and FSH
ß-subunits (4).
Analysis of
-subunit promoter activity has led to the identification
of multiple cis-acting elements that interact to regulate
pituitary and gonadotrope-specific expression. As might be expected,
several of these elements appear to be conserved in the promoters of
-subunit genes across multiple species. These elements include a
binding site for a LIM (lin-11, isl-1, mec-3)-homeodomain protein (5, 6), which cooperates with two regulatory proteins that bind to
immediately adjacent elements (7); several canonical E-boxes (8); the
ACT element that binds members of the GATA binding factor family
(9); and the gonadotrope-specific element or GSE that binds the nuclear
orphan receptor, steroidogenic factor-1 (SF-1) (10, 11). In addition to
these elements, the human glycoprotein hormone
-subunit gene also
contains tandem cAMP-responsive elements (CREs) that are important for
basal promoter activity not only in the pituitary but also in placenta,
a normal site of expression of the
-subunit gene in primates
(12, 13, 14).
While considerably less is known regarding the organization of
regulatory elements in the promoters for the gonadotropin ß-subunit
genes, functional homologs of the
-subunit GSE have been identified
in the proximal promoter of both the rat (15) and bovine LH ß-subunit
genes (16). More recently, we have identified a GSE homolog located at
-245 to -237 bp in the murine GnRH-R gene promoter (17). This GSE
homolog, as with those in the
- and LH ß- subunit genes, is
capable of binding SF-1, and mutation of this element leads to
approximately 60% loss of promoter activity in
T31 cells. Thus,
the conserved function of the GSE in the common
, LHß, and GnRH-R
genes suggests that SF-1, or a very similar protein, serves a common
role in regulating expression of multiple gonadotrope-specific
genes. Consistent with this regulatory role is the restricted
expression of SF-1 to gonadotrope cells in the anterior pituitary gland
(18). However, SF-1 is also expressed in a number of extrapituitary
sites, most prominently in steroidogenic tissues including the ovaries,
testes, and adrenal glands (19, 20). Consequently, while the GSE is a
key element in the murine GnRH-R gene promoter, the nonpituitary
expression of SF-1 suggests to us that this element alone may not be
the key mediator of cell-specific expression.
In this regard, we have used a combination of deletion and mutational
analyses to define multiple elements in the proximal promoter of the
murine GnRH-R gene. Of particular importance to basal activity in
T31 cells were elements residing within the proximal 500 bp of
5'-flanking sequence and, more specifically, in the region residing
between -500 and -400 bp relative to the start site of translation
(21). A functional scan of this 100-bp region by block replacement
mutagenesis revealed the presence of two elements that contribute to
basal activity of the murine GnRH-R promoter in
T31 cells (17).
First, mutation of the region residing between -482 and -475 bp
resulted in loss of approximately 25% of promoter activity. More
importantly, however, was a 58% reduction in promoter activity upon
mutation of the sequence between -393 and -386 bp. We have termed
this more proximal element the GnRH-R-activating sequence (GRAS). Based
on these studies, we proposed a model in which cell-specific activity
of the murine GnRH-R gene promoter is mediated by a complex enhancer
that includes, but is not limited to, a binding site for SF-1 and an
element located at approximately -393 bp (17). Remaining at issue,
however, is whether these elements fully account for basal activity of
the murine GnRH-R promoter in
T31 cells or whether additional
elements contribute to activity of the GnRH-R promoter. In particular,
we were interested in addressing the potential contribution of a
canonical activator protein 1 (AP-1) site (TGAGTCA) (22) located at
-336 to -330 bp. Accordingly, in the current studies combinatorial
mutagenesis was used to assess the relative contribution of multiple
cis-acting elements to GnRH-R promoter function. Herein, we
report that basal activity of the proximal promoter of the murine
GnRH-R gene is dependent on an SF-1-binding site, an AP-1 element, and
a novel element, termed GRAS, that is capable of conferring
cell-specific activity on a heterologous, minimal promoter.
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RESULTS
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Multiple Elements Contribute to Basal Activity of the Murine GnRH-R
Gene Promoter
To address the relative contributions of the SF-1-binding site,
GRAS, and AP-1 elements, block replacement vectors were constructed
that contained either a NotI (µSF-1, µGRAS) or
EcoRI (µAP-1) restriction site in place of the wild type
sequence. Each mutant was constructed in the context of 600 bp of
proximal promoter and tested for activity by transient expression in
T31 cells. Consistent with previous data (17), mutations in both
the SF-1-binding site (µSF-1) and GRAS (µGRAS) resulted in a
greater (P < 0.05) than 50% attenuation in
transcriptional activity (Fig. 1
).
Similarly, disruption of the canonical AP-1 element (µAP-1) at
position -336 bp reduced (P < 0.05) promoter activity
by 64% as compared with the wild type -600 promoter. An approximate
80% reduction (P < 0.05) in promoter activity was
noted in vectors containing double mutation combinations of either
µGRAS, µSF-1, or µAP-1. Finally, a vector containing mutations in
all three of these elements reduced promoter activity to a level not
different (P > 0.05) from the promoterless control
(LUC). Thus, we suggest that three elements: an SF-1-binding site, an
AP-1 site, and a noncanonical site located at -393 bp serve as the
major elements mediating basal activity of the proximal murine GnRH-R
promoter in
T31 cells.

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Figure 1. Three Major Elements Regulate Activity of the
Cell-Specific Basal Promoter of the GnRH-R Gene
Vectors containing mutations in a single element, two elements, or all
three elements were contransfected with pRSV-LacZ using LipofectAMINE
as described in Materials and Methods. At 24 h
posttransfection, cells were harvested and cellular lysates were
assayed for luciferase and ß-galactosidase activity. Luciferase
activity was adjusted for ß-galactosidase activity, and values are
expressed as the percent of pMGR-600LUC activity (1.15 ± 0.311,
mean ± SD). Values reported in the figure represent
the mean ± SD of triplicate samples in at least three
different transfections. a, b, c, d, Bars bearing different
letters are different (P < 0.05).
Unadjusted luciferase activity of pMGR-600LUC averaged 1297 ± 799
(mean ± SD).
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Functional Fine-Mapping of the Region from -393 to -376 bp
Outlines a New Element
To further refine the functional domain of GRAS, we generated a
series of mutant vectors in which 2-bp transversion mutations were
incorporated across the region from -395 to -378 bp (µGRAS-1 to
µGRAS-9; Fig. 2
). These mutant vectors
were transiently transfected into
T31 cells and compared with the
wild type promoter (pMGR-600LUC). Analysis of this series of mutations
outlined a core motif in which µGRAS-3 through µGRAS-8 each reduced
promoter activity by 56%, 64%, 68%, 43%, 61%, and 53%,
respectively, as compared with pMGR-600LUC (all values
P < 0.01; Fig. 2
). Thus, each of these 2-bp
transversion mutations reduced promoter activity by approximately the
same amount (63%) as the block replacement mutant, µGRAS.
Furthermore, the element outlined by these mutations was flanked on
either end by a mutation that did not significantly effect promoter
activity. Based on these data, the critical core motif of the GRAS
element consists of the sequence 5'-CTAGTCACAACA-3' located at -391 to
-380 bp. Finally, although µGRAS-1 also had significantly lower
activity than pMGR-600LUC, it was separated from the motif by a 2-bp
mutation that did not effect promoter activity and consequently was not
considered part of the core element.

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Figure 2. Functional Fine-Mapping of µ11 Reveals a New
Element
To define the functional limits of the GRAS element, a series of 2-bp
mutations were generated across the region from -395 to -378 in the
context of the wild type pMGR-600LUC vector. The wild type sequence is
shown across the top of the figure with the mutations in
lowercase letters below. These vectors were
cotransfected with RSV-LacZ in T31 cells using the lipofectamine
procedure as described in Materials and Methods. At
24 h posttransfection, the cells were harvested and assayed for
luciferase and ß-galactosidase activity. Luciferase values were
corrected for ß-galactosidase activity, and all values are expressed
as the percent of pMGR-600LUC activity. Values represent the mean
± SD of triplicate samples in at least three different
transfections. *, Vectors that are significantly different from
pMGR-600LUC (P < 0.01).
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GRAS Confers
T31 Cell-Specific Enhancer Activity upon a
Heterologous Promoter
Next we sought to determine whether GRAS was capable of acting as
a stand-alone enhancer of transcriptional activity and if so, whether
the activation was cell-specific. An oligonucleotide containing the
core motif plus four flanking bases at both the 5'- and 3'-ends, (5'
CTGTCTAGTCACAACAGTTT-3'), was used to generate a luciferase
expression vector in which two or three direct repeats of the element
were inserted upstream of the TK minimal promoter (3xGRAS-TKLUC). We
also constructed a similar vector with three direct repeats of the
element mutated as in µGRAS-5 (5'
CTGTCTAGGAACAACAGTTT 3'; 3xµGRAS-TKLUC), to
verify that any transcriptional activation of 3xGRAS-TKLUC was due to
binding of a specific protein(s) to the GRAS elements and not an
artifact of vector construction. These vectors were transiently
transfected into
T31 cells. While two copies of GRAS 5' to TK led
to a modest 1.8-fold increase over TK (data not shown), addition of
three copies of GRAS led to an approximately 5-fold increase
(P < 0.01; Fig. 3
) in
luciferase expression as compared with the TK vector alone (TKLUC).
Consistent with the mutational analysis, the insertion of three copies
of µGRAS-5 did not lead to any enhancement of luciferase expression
as compared with TKLUC (P > 0.05) (Fig. 3
).

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Figure 3. The GRAS Element Acts as a Stand-Alone Enhancer
3xGRAS-TKLUC and 3xµGRAS-TKLUC were cotransfected with RSV-LacZ into
T31 cells using the lipofectamine procedure as described in
Materials and Methods. At 24 h posttransfection,
the cells were harvested and assayed for luciferase and
ß-galactosidase activity. Luciferase values were corrected for
ß-galactosidase activity, and all values are expressed as fold over
the negative control TKLUC. *, Values significantly different from
TKLUC P < 0.01.
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To test whether the enhancer activity of 3XGRAS was confined to cells
of gonadotrope origin, the 3xGRAS-TKLUC vector was transiently
transfected into the
T31 cell line, Cos-7 kidney epithelial cells,
GH3 pituitary somatotrope-derived cells, HeLa cervical carcinoma cells,
BeWo human choriocarcinoma cells, and MA-10 Leydig tumor cells. As in
the previous experiment, 3xGRAS-TKLUC activity was 6-fold greater
(P < 0.001) than TKLUC in
T31 cells and 1.43-fold
greater (P < 0.001) than TKLUC in BeWo cells (Fig. 4
). Although the stimulation in BeWo
cells was much less than that of the
T31 cell line, since GnRH-Rs
are expressed in the human placenta (23, 24), GRAS may represent a
mechanism underlying regulation of GnRH-Rs in the placenta. In
contrast, three copies of GRAS had no effect (P >
0.05) on TKLUC activity in Cos-7, HeLa, GH3, or MA-10 cells.

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Figure 4. GRAS Has Enhancer Activity Only in the T31
Cell Line
3xGRAS-TKLUC and pRSV-LacZ were cotransfected into T31, HeLa,
Cos-7, MA-10, BeWo, and GH3 cells as described in Materials and
Methods. At 24 h posttransfections, the cells were
harvested and assayed for luciferase and ß-galactosidase activity.
Luciferase values were normalized for ß-galactosidase expression.
Promoter activity is expressed as fold of TKLUC expression. ***, Values
significantly different from TKLUC P < 0.001.
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DISCUSSION
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The restricted expression of a set of genes to a specific cell
type provides an opportunity to dissect the common and unique pathways
directing this cell-specific expression. Gonadotrope cells of the
anterior pituitary gland are characterized by their expression of the
common
-subunit and specific ß-subunits of the LH and FSH
glycoprotein hormones, and the GnRH-R. The emerging model for
gonadotrope expression of the
-subunit establishes that, as in
placenta, tandem cAMP response elements (CREs) are critical for basal
promoter activity (10, 25, 26), although different members of the
CRE-binding protein/activating transcription factor family may bind
these elements in trophoblasts and gonadotropes (14). In addition, four
other elements contribute to gonadotrope expression. These elements are
the GSE, the pituitary glycoprotein hormone basal element, and the
-basal elements 1 and 2 (5, 6, 7, 10, 11). These elements interact both
additively and synergistically to create a complex combinatorial code
that is responsible for expression of the common
-subunit in
gonadotropes (7). Such an arrangement of multiple elements into complex
enhancers that act to confer tissue/cell-specific expression has
emerged as a hallmark of multiple genes (27, 28, 29). Thus, it seems likely
that a complex enhancer may also mediate gonadotrope-specific
expression of the LH ß-subunit and the GnRH-R. Although much less is
known about the specific elements, there has been progress in recent
years toward defining functional regions in the promoters of the LHß
and GnRH-R genes. Of particular interest is the presence of functional
SF-1-binding sites in both the LHß (15, 16) and the GnRH-R (17)
genes. Therefore, among these genes expressed in gonadotropes, there
appear to be both common and unique elements regulating gene
expression. Such a combinatorial code allows both coordinated
stimulation of the gene group as well as differential regulation of
individual genes.
In the current study, we employed the gonadotrope-derived
T31 cell
line as a model to further dissect the elements responsible for
cell-specific basal activity of the GnRH-R gene promoter. We propose a
model in which cell-specific basal activity is regulated by a
tripartite enhancer composed of, but not limited to, binding sites for
SF-1, AP-1, and a novel element we have termed GRAS. This model is
based on several lines of evidence. First, the 250-bp region from -492
to -235 bp is capable of conferring full, cell-specific basal activity
upon a heterologous minimal promoter (17). Second, within that 250-bp
region we used block replacement mutagenesis to functionally define
both the SF-1-binding site and the element located between -393 and
-376 bp (GRAS) (17). Finally, in the present study, we have also
identified a functional role for a canonical AP-1 element located at
-336 to -330 bp. The individual mutation of each of these three
elements results in a loss of approximately 60% of promoter activity,
while the mutation of all three elements reduces luciferase expression
to a level not significantly different from the promoterless luciferase
vector. Although these data do not obviate the existence of other
elements that may contribute to activity of the wild type promoter, it
does suggest that these three elements are major contributors to basal
promoter activity. Furthermore, since each of the three combinations of
double mutations results in a nearly identical decrease in promoter
activity, each individual element may contribute equally to promoter
function and act independently of the other elements. This is in marked
contrast to the
-subunit promoter, in which mutation of a single
element, the tandem CREs, abrogates placental expression (30) and
accounts for a greater than 80% reduction of promoter activity in
pituitary cell lines (7).
A question arises in regard to the tripartite cell-specific enhancer of
the GnRH-R gene. Specifically, is full, basal cell-specific activity
due to a unique combination of elements or a single cell-specific
element? In regard to the former, we have shown that mutation of any of
the three elements that comprise the cell-specific enhancer
significantly decreases promoter activity. Thus, full activity of the
GnRH-R gene promoter is undoubtedly due to the unique combination of
the AP-1 element, SF-1-binding site, and GRAS element. The question
regarding a single cell-specific element is more complicated. At first
glance it would be easy to dismiss both SF-1 and AP-1 as elements
regulating cell-specific GnRH-R promoter activity because of the
expression of SF-1 in cells other than gonadotropes and the ubiquitous
expression of members of the Jun/Fos family of transcription factors.
However, upon further consideration, these elements can not be excluded
from a role as cell-specific regulators. For example, while SF-1 is
expressed in tissues outside of the pituitary gland (19, 20), it must
be noted that among the cells of the pituitary gland, SF-1 expression
is restricted to gonadotropes (18). Similarly, AP-1 elements are bound
by a family of factors whose relative expression may differ among
tissues and that may interact with other non-Jun/Fos proteins that are
potentially cell-specific (31, 32, 33, 34, 35). In fact, the tandem CREs of the
-subunit gene promoter provide an excellent example of a situation
in which a common element serves as a cell-specific regulator by
binding to different members of the CRE-binding protein/activating
transcription factor family in trophoblast and gonadotrope cell lines
(14).
Regardless of how we view SF-1 and AP-1, the evidence for GRAS as a
cell-specific enhancer is clear. Three copies of the wild type GRAS
element (5'-CTAGTCACAACA-3') led to a 5- to 6-fold stimulation in
transcriptional activity of the TK minimal promoter in
T31 cells,
whereas three copies of the µGRAS-5 mutant element had no effect.
Furthermore, this enhancer activity of GRAS is not specific to the TK
promoter as 3xGRAS leads to a 22-fold stimulation of the rat PRL
minimal promoter in transient transfections of
T31 cells (data not
shown). The larger relative stimulation apparent with the PRL minimal
promoter (positions -33 to +13) is probably due to significantly lower
basal activity of the PRL promoter as compared with the TK promoter
(36). Finally, transcriptional activity of the 3xGRAS-TKLUC vector was
virtually unchanged when transiently transfected in a variety of other
cell types, including cells from both reproductive and nonreproductive
tissues. Since these cell types included the GH3 pituitary somatotrope
cell line, the factor(s) binding to the GRAS element are apparently not
common to all pituitary cell types. Hence, these data establish the
ability of GRAS to function as a stand-alone enhancer that stimulates
promoter activity specifically in a gonadotrope-derived cell line. As
such, the protein(s) binding to the GRAS element may be unique to
gonadotropes. In fact, we have previously shown sequence-specific
binding of protein(s) in
T31 cell nuclear extracts to the region
from -410 to -363, which contains the GRAS element. In contrast, no
binding activity to this region was detected using nuclear extracts
from Cos-7 cells (21).
In summary, we have identified a tripartite enhancer comprised of an
AP-1 element, a binding site for SF-1, and a novel element (GRAS) that
regulates basal activity of the GnRH-R gene promoter in the
gonadotrope-derived
T31 cell line. Finally, we propose that GRAS
may be the key component regulating this cell-specific basal enhancer.
Given the ability of the proximal promoter of the GnRH-R gene to direct
pituitary-specific expression of luciferase in transgenic mice (37),
perhaps the ultimate test of this hypothesis will be to test the
activity of a promoter containing a mutation of the GRAS element in
transgenic mice.
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MATERIALS AND METHODS
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Vector Construction
The construction of pMGR-600LUC, µGRAS, and µSF-1 has
been described (17, 21). The block replacement mutation of the AP-1
site located at -336 was generated by PCR amplification using a pair
of sense and antisense primers that replaced the AP-1 site with a
restriction site for EcoRI (GAATTC). These primers included
sufficient 5'- and 3'-flanking sequence (
18 bp) to assure specific
annealing. Overlapping fragments were generated from pMGR-600LUC
template DNA in separate PCR reactions using a sense primer from the
luciferase vector (RV3, Promega, Madison, WI) and the antisense mutant
primer or an antisense primer from the luciferase vector (GL2, Promega)
and the sense mutated primer. These products were combined and
amplified in a second round of PCR using the RV3 and GL2 primers. The
PCR product was digested with KpnI and XhoI and
inserted into the basic luciferase vector (pGL3-basic, Promega)
digested with the same enzymes.
The series of 2-bp transversion mutations of PMGR-600LUC were generated
using the same protocol. Sense and antisense mutant primers were
designed with 2-bp transversion mutations spanning the region from
-395 to -378 (5'-CTGTCTAGTCACAACAGT-3'; Fig. 2
). The correct size of
the resulting PCR products was determined by gel electrophoresis, and
the products were digested with KpnI and XhoI and
ligated into the vector fragment of pGL3-basic cut with the same
enzymes.
Multiple copies of the GRAS element were concatamerized by
phosphorylating one strand of a synthesized oligonucleotide containing
the element using polynucleotide kinase, annealing the two synthesized
strands, and ligating the mixture. Concatamers were subsequently
ligated into pBluescript SK (pBSK) digested with SmaI. The
construction of triple directionally inserted elements was verified by
sequencing. The 3XGRAS construct was subsequently digested out of the
pBluescript vector using XbaI and XhoI
restriction endonucleases and ligated into TKLUC digested with the
NheI and XhoI restriction endonucleases.
Insertion of the TK minimal promoter (positions -105 to +51 bp) in the
luciferase expression vector was described previously (17). The
identity of all plasmids was verified by restriction enzyme digestion
and sequencing.
Cell Culture and Transient Transfections
All cell cultures were maintained in a humidified
atmosphere of 5% CO2 at 37 C.
T31 cells were cultured
in high-glucose DMEM containing 2 mM glutamine, 5% FBS,
5% horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin
sulfate. Cos-7, GH3, and HeLa cells were maintained in high-glucose
DMEM containing 2 mM glutamine, 10% FBS, 100 U/ml
penicillin, and 100 µg/ml streptomycin sulfate. BeWo cells were
cultured in Waymouths media containing 15% FBS, 100 U/ml penicillin,
and 100 µg/ml streptomycin sulfate. MA-10 cells were cultured in RPMI
1640 media containing 2 mM glutamine, 15% horse serum, and
50 µg/ml gentamicin. GH3 cells were transfected using a calcium
phosphate/DNA coprecipitation method as previously described (30)
followed at 12 h after transfection by a 4-min exposure to 15%
dimethyl sulfoxide and 10% FCS in DMEM. After the dimethyl sulfoxide
shock, cells were washed twice with PBS, fresh media were added, and
the cells were incubated for 48 h before harvest.
T31, Cos-7,
BeWo, MA-10, and HeLa cell cultures were transfected using the
LipofectAMINE procedure (GIBCO/BRL Life Technologies, Gaithersburg, MD)
as previously described (21). Briefly, on the day before transfection,
0.51.5 x 106 cells were seeded into 35-mm wells of
a six-well tissue culture plate. Transfections included 1.4 µg test
vector and 0.25 µg pRSV-LacZ with 5 µl lipofectAMINE reagent (0.5
µg in 10 µl for BeWo and MA-10 cultures) in 200 µl high-glucose
DMEM containing 2 mM glutamine. This mixture was incubated
at room temperature for 30 min, then diluted to 1 ml with the same
media and applied to cell cultures previously washed with high-glucose
DMEM containing 2 mM glutamine. The cell cultures were
incubated with the transfection mixture for approximately 16 h at
37 C in a humidified atmosphere of 5% CO2. After
incubation,
T31, Cos-7, and HeLa cells were supplemented with 1 ml
high-glucose DMEM containing 2 mM glutamine, 10% FBS, 10%
horse serum, and antibiotics. In cultures of BeWo or MA-10 cells, the
transfection medium was aspirated and 2 ml of their respective culture
medias were added. After 56 h, media were aspirated, and cells were
washed twice with ice-cold PBS (pH 7.4). Cells were lysed in the wells
by the addition of 200 µl lysis buffer (0.025 M
glycylglycine, pH 7.8, 1 mM dithiothreitol, 15
mM MgSO4, and 1.0% Triton X-100). Cellular
debris was removed from lysate by microcentrifugation at 16,000 x
g for 2 min. Lysates were immediately assayed for luciferase
activity by adding 20 µl lysate to 100 µl luciferin substrate
(Promega) and measuring luminescence with a Turner model TD-20E
luminometer set for a 5-sec delay and 10-sec integration.
ß-Galactosidase activity was measured in 50 µl lysate using the
luminescent assay system and substrate (Tropix, Bedford, MA) with the
same luminometer set for 10-sec delay and 5-sec integration following
manufacturers instructions. Luciferase activity was normalized for
transfection efficiency by dividing the luciferase activity by
ß-galactosidase activity. Each vector was transfected in triplicate
for each transfection, and transfections were repeated at least three
times using two to three different plasmid preparations.
Statistical Analysis
The transfection data were analyzed by one-way ANOVA with vector
as the independent variable. If the F test was significant
(P < 0.05), means were separated using Tukeys (Fig. 1
) or Dunnetts (Figs. 2
, 3
, and 4
) methods of multiple comparisons
(38).
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FOOTNOTES
|
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Address requests for reprints to: Colin M. Clay, Animal Reproduction and Biotechnology Laboratory-Foothills Campus, Colorado State University, Fort Collins, Colorado 80523.
This work was supported by NIH Grant R29HD-32416. D.L.D. was supported
by NIH Postdoctoral Fellowship NRSA 1F32HD08169.
Received for publication June 10, 1997.
Revision received August 1, 1997.
Accepted for publication August 15, 1997.
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