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From the * Department of Cell Biology and
Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas;
and the Department of Cell and Developmental
Biology, Vanderbilt University School of Medicine, Nashville, Tennessee.
| Correspondence to: Dr Gail A. Cornwall, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th St, Lubbock, TX 79430 (e-mail: gail.cornwall{at}ttuhsc.edu). |
| Received for publication August 19, 2004; accepted for publication November 4, 2004. |
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Abstract |
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Key words: Epididymis, testis, spermatogenesis, gene regulation
Cres also differs from the cystatins by exhibiting tissue-specific expression. In contrast to the ubiquitous expression of cystatin C, Cres messenger RNA (mRNA) is present in the principal cells of the proximal caput epididymis, round spermatids, and anterior pituitary gonadotropes and was found recently in the ovary (Cornwall et al, 1992; Cornwall and Hann, 1995; Sutton et al, 1999; Hsia and Cornwall, 2003). Furthermore, Cres expression in the epididymis is dependent on testicular factors (Cornwall et al, 1992). To identify transcriptional elements necessary for the cell- and tissue-specific expression of the Cres gene, we initiated studies to examine the Cres gene promoter. Previously, an analysis of 1.6 kilobases (kb) of the mouse Cres promoter revealed transcription factor binding motifs present in highly regulated genes (Cornwall et al, 1999). These predicted DNA-binding elements included GATA, SF-1 (steroidogenic factor 1), SRY (sex-determining region Y), ERE (estrogen responsive element), Ptx1 (pituitary homeobox 1), and C/EBP (CCAAT/enhancer binding protein). We recently demonstrated that the transcription factor C/EBPß is the predominant C/EBP family member in the mouse epididymis and gonadotroph cells and is required for maximal expression of the Cres gene in these tissues (Hsia and Cornwall, 2001).
The purpose of this study was to determine whether 1.6 kb of Cres 5'-flanking sequences contain the necessary elements for cell- and tissue-specific expression. Toward this end, transgenic mice were generated that expressed the chloramphenicol acetyltransferase (CAT) gene under the control of a mouse Cres promoter fragment.
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Materials and Methods |
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Chimeric Construct
A DNA fragment containing approximately 1.6 kb of Cres promoter
was generated by PCR with Cres 5'-flanking pGEM-T plasmid as
template (Cornwall et al,
1999). The DNA fragment was purified on a 1% agarose gel and
ligated into the promoterless pBLCAT3 plasmid (containing the bacterial CAT
sequence) by standard methods such that the Cres promoter DNA
fragment would drive expression of the CAT reporter gene. The 1.6-kb
Cres-pBLCAT3 plasmid was digested with appropriate enzymes to remove
vector sequences. The digested DNA was electrophoresed on a 1% low-melting
point agarose gel in 1x Tris-acetate EDTA (TAE) buffer (Invitrogen,
Carlsbad, Calif) for several hours, and the Cres-CAT cassette was
excised and purified with GELase (Epicentre, Madison, Wis). The DNA pellet was
washed several times in 70% ethanol before resuspending in Tris-EDTA (TE)
buffer, pH 7.5. The A260/280 was determined, and a small aliquot
was run on a 1% agarose/TAE gel to determine the DNA quality before pronuclear
DNA microinjection.
Transgenic Mice and DNA Isolation
Transgenic mice (strain B6D2, Harlan Sprague-Dawley) were generated by
microinjection of the linearized 1.6-kb Cres-CAT DNA into the male
pronucleus of fertilized oocytes with the use of standard techniques
(Palmiter and Brinster, 1985).
Transgenic animals were identified by genomic Southern blot analysis.
Approximately 3 to 4 mm of the tails from weaned mice was digested overnight
at 55°C in 210 µL of digestion solution (50 mM Tris-Cl, pH 8, 100 mM
NaCl, 100 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1.0 mg/mL of proteinase
K). The DNA was extracted with 1 volume of Tris-saturated phenol, followed by
1:1 (vol/vol) phenol/chloroform, and then twice with chloroform. The DNA was
subsequently precipitated at room temperature with 2.5 volumes of 100%
ethanol. Samples were centrifuged at 10 000 x g at 4°C for
15 minutes, washed with 70% ethanol, and dried briefly at room temperature.
The pellets were gently resuspended in 100 µL of 0.1x saline-sodium
citrate (SSC) and allowed to dissolve overnight at 4°C.
Southern Blot Analysis
Genomic DNA (10 µg) prepared from tail snips was digested overnight with
40 U of PstI restriction enzyme (Invitrogen) and electrophoresed
overnight on a 0.8% agarose/1x TAE gel. The gel was incubated in 0.25 N
HCl for 20 minutes, denatured in 0.5 M NaOH and 1.5 M NaCl for 45 minutes, and
washed in 0.5 M Tris, pH 8, and 1.5 M NaCl for 45 minutes. The DNA was then
transferred by vacuum blotting (Appligene, Illkirch, France) to nylon membrane
(Nytran Supercharge, Schleicher and Schuell, Keene, NH). The membrane was
ultraviolet (UV) cross-linked (Stratalinker; Stratagene, La Jolla, Calif) and
prehybridized in Church buffer (0.5 M NaPO4, pH 7.5, 1 mM EDTA, 7%
SDS) at 65°C for 2 hours. Random-primed 32P-labeled probes were
synthesized from a 500-bp Cres promoter insert or the CAT insert
(Prime It II kit; Stratagene) and were incubated overnight with the membrane
(106 cpm/mL) in Church buffer at 65°C. The membrane was washed
twice for 20 minutes at room temperature in 0.2x SSC and 0.1% SDS before
exposure to autoradiographic film. Transgene copy number was determined from
the ratio of the intensity of the DNA fragment corresponding to the transgene
and the 7-kb PstI fragment corresponding to the endogenous
Cres gene multiplied by 2. Mice homozygous for the CAT transgene were
identified from those heterozygous for the transgene by comparing the relative
band intensities following Southern blot analysis.
CAT Enzyme-Linked Immunosorbent Assay
The amount of CAT protein present in different tissues from transgenic mice
heterozygous and homozygous for the Cres-CAT transgene was measured
with a CAT enzyme-linked immunosorbent assay (ELISA; Roche Molecular
Biochemicals, Indianapolis, Ind). Briefly, small tissue fragments from several
mice were pooled and homogenized in 1x lysis buffer provided by the kit
and protein concentrations were determined by bicinchoninic acid assay
(Pierce, Rockford, Ill). CAT protein standards and 150 µg of each tissue
lysate were added to the microplate and incubated for 2 hours at 37°C. The
wells were washed 5 times with washing buffer provided in the kit and then
incubated for 1 hour at 37°C with anti-CAT antibody conjugated to
digoxigenin (anti-CAT DIG). The wells were washed again with buffer and
incubated with anti-DIG antibody conjugated to peroxidase (anti-DIG POD) for 1
hour at 37°C. Sample wells were washed with washing buffer and incubated
with peroxidase substrate and enhancer and the absorbance at 405 nm was
measured with a Bio-Tek EL 312e microplate reader (Bio-Tek Instruments Inc,
Winooski, Vt). Data (pg CAT protein/100 µg of tissue) are presented as the
mean ± SEM of 3 independent experiments.
Reverse Transcription-PCR
Total RNA was isolated from mouse tissues with Trizol reagent (Invitrogen)
following the manufacturer's protocol. The RNA was quantitated by
A260/A280 and visualized by gel electrophoresis in 1%
agarose gel containing 1x borate buffer, pH 8.2, and 0.66 M formaldehyde
with ethidium bromide in the RNA samples. For reverse transcription (RT)-PCR,
2.5 µg total RNA was incubated in RT reaction buffer containing 5 mM
MgCl2, 50 mM KCl, 10 mM Tris, pH 8.3, 0.5 mM desoxynucleotide
triphosphates (dNTPs), 20 U RNasin (RNase inhibitor, Promega, Madison, Wis),
and 2.5 µM oligo-dT (Promega) in a final volume of 25 µL for 30 minutes
at 37°C in the presence of 2.5 U RNase-free DNase I (Roche Molecular
Biochemicals, Indianapolis, Ind). After heat inactivation of DNAse I at
75°C for 5 minutes, an aliquot was reserved for PCR amplification as a
no-RT control to confirm the absence of contaminating DNA. Fifty units of MuLV
reverse transcriptase (Perkin-Elmer Biosystems, Foster City, Calif) was added
to the remainder and reverse transcription was carried out at 42°C for 30
minutes, 99°C for 5 minutes, and 4°C for 5 minutes.
Three microliters each of RT and no-RT reaction was amplified by PCR in separate reactions with primers recognizing CAT, Cres, and S16 complementary DNAs (cDNAs). S16 was amplified as a constitutive control to measure the relative efficiency of each RT reaction. The identity of the PCR products generated with each primer pair was confirmed by sequence analysis. PCR master mixes containing 10 mM Tris, pH 8.3, 50 mM KCl, 0.5 µM each of forward and reverse primers, and 1.25 U of Taq DNA polymerase (Sigma Chemical Co, St Louis, Mo) were prepared so that RNA samples within a particular experiment were amplified from a single master mix. MgCl2 and dNTP concentrations, as well as cycle number, were optimized for each set of primers. Cres PCR reactions were carried out in 2.5 mM MgCl2 and 0.25 mM dNTPs for 25 (reproductive tract samples) or 40 (pituitary samples) cycles. CAT and S16 reactions were amplified with 2 mM MgCl2 and 0.2 mM dNTPS for 40 and 26 cycles, respectively. The cycling parameters consisted of 45 seconds at 95°C for denaturation, 25 seconds at annealing temperature (Ta) for each primer set, and 1 minute at 72°C for extension, after which the reactions were incubated at 72°C for 7 minutes with a minicycler (MJ Research Inc, Watertown, Mass). RT-PCR products were analyzed by gel electrophoresis in 1.5% agarose/1x TAE gels followed by ethidium bromide staining and examination under UV light. Each animal experiment was repeated, and in each case, the results of a representative experiment are shown.
Oligonucleotide Primer Pairs
PCR primers (Invitrogen) were designed from the sequences for mouse CAT,
Cres, and S16 cDNAs with the use of PrimerSelect (Lasergene Suite;
DNA Star, Madison, Wis) as follows: Cres sense:
5'-CAAGGAAAGTGAGGACAAATATGTC-3' and antisense:
5'-GTGACAGACTTGAACCACAGGTT-3', Ta = 64°C,
25 or 40 cycles; CAT sense: 5'-TCTTGCCCGCCTGATGAATGCTC-3' and
antisense: 5'-TACGCCCCGCCCTGCCACTC-3', Ta =
56°C, 40 cycles; and S16 sense: 5'-CGCTGCAGTCCGTGCAGGTCTT-3'
and antisense: 5'-TCCAAACTTTTTGGATTCGCAGCG-3',
Ta = 56°C, 25 cycles.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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CAT Protein Expression in Transgenic Mouse Tissues
We previously showed that the Cres gene is expressed in the
principal cells of the proximal caput epididymis, round spermatids in the
testis, anterior pituitary gonadotropes, and, most recently, corpora lutea of
the ovary (Cornwall et al,
1992; Cornwall and Hann,
1995; Sutton et al,
1999; Hsia and Cornwall,
2003). To determine whether the CAT transgene was expressed in
mouse tissues in a similar tissue-specific manner, a CAT ELISA was used to
measure CAT protein in tissue extracts from different regions of the mouse
epididymis, as well as in 16 other tissues from male and female mice
heterozygous and homozygous for the transgene. As shown in
Figure 2, CAT protein exhibited
tissue-specific expression similar to that of endogenous CRES and was detected
in mouse testis and the distal regions of the epididymis; however, the highest
amount of CAT protein was present in spermatozoa isolated from the cauda
epididymis. Because of variations within the CAT assay, however, CAT protein
levels in spermatozoa were not statistically different from that in the
testis. Lower levels of CAT protein were present in the proximal regions of
the epididymis, vas deferens, female reproductive tract, and pituitary gland.
CAT protein was not detected in the seminal vesicle, prostate, brain, heart,
liver, kidney, spleen, adrenal, and lung of transgenic mice. In general, mice
homozygous for the transgene had approximately twice the levels of CAT protein
present in mice heterozygous for the transgene, suggesting a proportional
increase as the result of an increase in copy number. One exception to this
was in the female anterior pituitary gland, in which there appeared to be
higher levels of CAT protein in the heterozygous animals compared with the
homozygous animals. Also, CAT protein appeared to be higher in female anterior
pituitary glands compared with male glands. Because in separate studies we
have determined that Cres mRNA and protein in the anterior pituitary
gland are differentially regulated by gonadotropin-releasing hormone (GnRH)
and steroid hormones (Sutton-Walsh and Cornwall, unpublished observations),
the differences we see in CAT protein levels likely reflect the hormonal state
of the animals rather than true sex differences or differences between
heterozygous and homozygous mice. As expected, tissues isolated from control,
nontransgenic mice had no detectable CAT protein (data not shown). The high
amount of CAT protein in the testis and in isolated cauda spermatozoa, as well
as the increasing amounts of CAT protein along the epididymal tubule, strongly
suggested that the 1.6-kb Cres promoter possessed the necessary
elements to confer expression of the CAT reporter in the testis and,
specifically, in the germ cells. However, at this time, we could not rule out
that the epididymal epithelium also expressed CAT protein.
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RT-PCR Analysis of CAT mRNA in Transgenic Mouse Tissues
RT-PCR was carried out to examine Cres and CAT mRNA expression in
the testis and 5 regions of the mouse epididymis from mice homozygous for the
Cres-CAT transgene. Previously, we demonstrated by Northern blot
analysis that expression of the Cres gene was highest in the proximal
caput (region 1), with lower expression in the midcaput (region 2) and testis
(Cornwall et al, 1992). RT-PCR
confirmed our initial studies and showed Cres mRNA in the testis and
epididymal regions 1 and 2 (Figure
3). In contrast, CAT mRNA was only detected in the testis and not
the epididymis, suggesting that the CAT protein detected along the epididymal
tubule and in the vas deferens of CAT transgenic mice was from spermatozoa. We
cannot eliminate the possibility, however, that CAT mRNA is present in these
tissues, but at very low levels.
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We also used RT-PCR to examine Cres and CAT mRNA expression in the female reproductive tract and pituitary gland from male and female transgenic mice (Figure 4). As expected, Cres mRNA was present in the ovary, with lower levels in both male and female pituitary glands. Because Cres mRNA in the pituitary gland is hormonally regulated (Sutton-Walsh and Cornwall, unpublished observations), the differences in Cres mRNA levels between the pituitaries from males and females likely reflect the hormonal state of the animals. Interestingly, low levels of Cres mRNA were also detected in the oviduct and uterus, revealing new sites of Cres expression not previously detected by our Northern blot studies (Cornwall et al, 1992). CAT mRNA was detected in the male and female pituitary gland but not in the female reproductive tissues, suggesting that, in addition to driving transgene expression in the testis, the 1.6-kb Cres promoter fragment can also drive pituitary gland expression. Whether the 1.6-kb promoter fragment also contains all the necessary elements for the same response to hormonal stimuli as the endogenous Cres gene in the pituitary gland is not known. The higher levels of CAT mRNA in the male compared with the female pituitary, which is opposite that of Cres mRNA levels, might suggest subtle differences between the transgene and the endogenous Cres gene in their response to hormones. Similar to our observations in the epididymis and vas deferens, we cannot rule out that CAT mRNA is present, but at very low levels, in female reproductive tissues.
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Temporal Expression of CAT mRNA in the Testes of Postnatal Mice
The mRNA and protein analyses thus far suggested that the CAT transgene was
expressed by the testicular germ cells. We previously established by in situ
hybridization that Cres mRNA is primarily expressed by round and
elongating spermatids (Cornwall and Hann,
1995). However, because CAT mRNA and protein levels were low in
the transgenic mice, we were unable to use in situ hybridization or
immunohistochemistry to determine whether the CAT transgene was expressed in
the same germ cell populations as the endogenous Cres gene. An
indirect approach to look at expression during different stages of male germ
cell development was to examine CAT mRNA levels in testes from mice at
different postnatal ages, which reflected expression at different points along
the first wave of spermatogenesis. The first round of spermatogenesis in mouse
starts after birth and is characterized by the sequential appearance of cells
corresponding to each stage of spermatogenesis
(Bellve et al, 1977).
Spermatogonia typically appear at postnatal days 6 through 7 and round
spermatids at postnatal days 22 through 25. As shown in
Figure 5, CAT mRNA was first
detected in the testes from day 22 mice, which corresponded with the
appearance of Cres mRNA and with approximately the first appearance
of round spermatids. CAT and Cres mRNA were also detected in the
testes from mice at days 26 and 31, which likely reflected accumulation of
round spermatids and the appearance of elongating spermatids. Although we
cannot completely eliminate the possibility that CAT mRNA is expressed at
specific stages by Sertoli, Leydig, or other somatic cells in the testis, the
parallel temporal expression of Cres and CAT mRNAs in the postnatal
mice suggests that, like the endogenous Cres gene, CAT is present in
the round and elongating spermatids. These data also indicate that the 1.6-kb
Cres promoter fragment has sufficient regulatory sequences to
recapitulate the developmentally regulated expression of the endogenous
Cres gene in the testis.
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Discussion |
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In addition to our studies presented here, several studies have identified spermatid-specific gene promoters. It is of interest that many of these promoters have been localized to short proximal promoter sequences, including protamine 1 (119 bp), t-ACE (91 bp), proenkephalin (119 bp), Phda-2 (187 bp), and Ldhc-4 (100 bp) (Howard et al, 1993; Zambrowicz et al, 1993; Iannello et al, 1997; Liu et al, 1997; Li et al, 1998). A comparison of several of these promoters with the Cres promoter revealed no similar stretches of sequence homology. Although a common motif is not present in all spermatid-specific promoters, there is a commonality of putative transcription factor binding motifs (Figure 6A), which could have important consequences for germ cell-specific expression.
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Another approach to identifying regions of sequence important for cell-specific expression is the comparison of gene promoters from different species. Previously, we sequenced portions of the rat and human Cres 5'-flanking sequences, and their alignment with the mouse Cres promoter sequence is shown in Figure 6B. We have also established that, like mouse and rat, human Cres mRNA localizes to the round spermatids (Wassler et al, 2002); therefore, promoter elements that are common to mouse, rat, and human could be critical for round spermatid-specific expression. In Figure 6B, regions of the 5'-flanking sequence that have high identity between all 3 species, and thus might bind transcription factors important for germ cell expression, are indicated by boxes. Computer analyses for known transcription factor binding sites yielded no matches, suggesting that these regions of sequence could represent novel transcription factor binding sites involved in the germ cell-specific expression of Cres.
In contrast to the somewhat limited studies of germ cell promoters, gene promoters that target expression to the anterior pituitary gland, and specifically to gonadotroph cells, have been more fully characterized. The gonadotrope-specific element (GSE), which interacts with the SF-1 transcription factor (Keri and Nilson, 1996), is important for conferring gonadotrope-specific expression and has been shown to regulate the expression of several genes involved in steroidogenesis and reproductive function (Ingraham et al, 1994). In addition to SF-1, the transcription factor Egr-1 (early growth response protein-1) has been shown to be important for full LHß gene expression, possibly by a synergistic interaction with SF-1, as well as GnRH responsiveness (Halvorson et al, 1998; Tremblay and Drouin, 1999). Pitx1 (pituitary homeobox 1) transcription factor also plays a key role in normal gonadotrope function (Lanctot et al, 1999; Szeto et al, 1999). Studies of the proximal LHß promoter indicate that full activation of the LHß gene, including GnRH responsiveness, requires the functional interactions of SF-1, Egr-1, and Pitx1 (Tremblay and Drouin, 1999; Quirk et al, 2001). Our studies showed that the 1.6-kb Cres promoter fragment targeted transgene expression in the anterior pituitary gland, thereby recapitulating the expression of the endogenous Cres gene. Similar to the LHß gene promoter, the 1.6-kb Cres promoter fragment contains a GSE (SF-1) element as well as an Egr-1 and several Pitx1 elements, suggesting that these sites might also play necessary roles for Cres expression in gonadotroph cells. As a result of the low levels of CAT mRNA and protein expression in the pituitary glands of transgenic mice, however, we were unable to confirm transgene expression in the gonadotroph cells.
Compared with tissues such as the liver or kidney that showed no CAT protein, low levels of CAT protein were consistently detected in the proximal epididymis and female reproductive tissues. However, we did not detect the corresponding CAT mRNA. The lack of CAT mRNA could be because specific DNA elements necessary for the activation of epididymal and female reproductive tract transcription are not present in the 1.6-kb Cres promoter fragment. Alternatively, because some CAT protein was detected, perhaps the levels of CAT mRNA are too low to be detected, suggesting that tissue-specific enhancers are missing from the promoter fragment. In fact, CAT expression in the testis and pituitary gland from transgenic animals was much lower than that of the endogenous Cres gene, suggesting that additional cis-acting sequences, such as specific enhancer elements in the endogenous Cres promoter, might also be necessary for high levels of expression in these tissues. Finally, we cannot rule out the possibility that the lack of CAT expression in the epididymis and ovary is because the transgene inserted into a region of the genome that prevented expression in these tissues.
It is of interest that, of the few transgenic studies that have been performed to identify promoter sequences that direct epididymal-specific expression, several have yielded inappropriate patterns of reporter gene expression or a complete lack of epididymal expression. The endogenous cysteine-rich secretory protein 1(CRISP-1) gene is expressed in the corpus and cauda regions of the mouse epididymis; however, 3.8 kb of the CRISP-1 5'-flanking sequence linked to EGFP (enhanced green fluorescent protein)-directed transgene expression to the testis rather than the epididymis (Lahti et al, 2001). In contrast to the expression of the endogenous glutathione peroxidase 5 (GPX5) gene in the caput region, a 5-kb promoter fragment of the gene directed expression of EGFP to a smaller region of the caput, as well as to the cauda epididymis and other mouse tissues (Lahti et al, 2001). Transgenic mice expressing 0.3-kb of the Pem homeobox gene promoter conferred transgene expression to the caput, where the endogenous Pem gene is expressed, but also an aberrant expression in the corpus. The aberrant expression was lost when 0.6 kb of Pem promoter was included in the transgene, suggesting that negative regulatory elements exist between 0.6 and 0.3 kb of the Pem promoter (Rao et al, 2002). To date, only 5 kb of the epididymal retinoic acid-binding protein (E-RABP) gene promoter and 5.3 kb of the related 17-kd murine epididymal protein (mEP17) gene promoter have been shown to drive reporter expression in an appropriate region- and epididymal-specific manner (Lareyre et al, 1999; Suzuki et al, 2003). Thus, it appears that the DNA elements that mediate region-specific gene expression in the epididymis are quite complex and will require further investigation to dissect out the critical components.
Taken together, our studies demonstrate that 1.6 kb of the mouse Cres promoter contains all the necessary information to drive the tissue-specific expression of the CAT transgene to the testicular germ cells and the anterior pituitary gland, thereby recapitulating, in part, the expression of the endogenous Cres gene. The 1.6-kb promoter fragment, however, lacks essential enhancer elements required for endogenous levels of expression of the reporter gene in these tissues. The Cres promoter fragment also lacks essential tissue-specific elements or enhancers that allow expression or normal levels of expression in the other Cres-expressing tissues, such as the epididymis and ovary. Further studies that use promoter deletion analyses are required to establish whether smaller Cres promoter fragments retain the ability to direct germ cell-specific expression of Cres. In addition, larger Cres promoter fragments are needed to identify regions that confer epididymal and ovarian expression.
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Acknowledgments |
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Footnotes |
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Current address: Karp Family Research Laboratories, 1 Blackfan Circle,
Floor 8; RB 08004B, Boston, MA 02115. 
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