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Male Reproductive Tract |
Department of Cell Biology3
Department of Internal Medicine,4 University of Virginia Health System, Charlottesville, Virginia 22908
Department of Cell Biology and Biochemistry,5 Texas Tech University Health Sciences Center, Lubbock, Texas 79430
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5' site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between –530 and –681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.
epididymis, gene regulation, male reproductive tract
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One potential method for analyzing initial segment-specific promoters is in vivo electroporation, which avoids the previously described pitfalls. Recently, in vivo electroporation has been used as a means of expressing genes in multiple tissues in the intact animal mostly to overexpress certain proteins [7–9]. Tissue-specific promoter analysis using this approach is a novel use of the in vivo electroporation method and has recently been used in muscle tissue [10]. In this study, the promoters of two genes, cystatin-related epididymal spermatogenic (cres) and -glutamyl transpeptidase (GGT), specifically expressed in the rodent initial segment, have been analyzed using in vivo electroporation and compared to previous results from in vitro analysis [11, 12].
In the mouse, cres expression is restricted to germ cells in the testis, anterior pituitary gonadotrophs, and the initial segment [13–15]. When the cres promoter (Fig. 1A) was tested in LTß2 gonadotroph cells, both 1.6 kilobases (kb) and 135 base pairs (bp) of promoter sequence were capable of reporter gene expression [11]. Within the 135-bp promoter sequence were two CCAAT/enhancer binding protein (C/EBP) DNA-binding sites [11]. Mutation of either C/EBP site reduced the expression of the reporter gene, and mutation of both sites essentially eliminated reporter gene expression [11].
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In the rat, there are five promoters that control the expression of five GGT mRNAs (I–V) [16]. Each mRNA contains a unique 5' untranslated region (UTR) and a common 5' UTR –144 bp upstream from the translation start site. The remainder of the mRNA is the same for all forms. Each mRNA encodes an identical protein, but each promoter controls the expression of GGT in a tissue-specific manner [16]. GGT mRNA IV is highly expressed in the rat initial segment [17, 18]. Previous studies were carried out to analyze 2 kb of promoter sequence for GGT mRNA IV in rat primary initial segment epithelial cell cultures [12]. This 2-kb promoter sequence contained several polyomavirus enhancer activator 3 (PEA3) DNA-binding sites (Fig. 1B). On cotransfection with PEA3, a 135-bp sequence was capable of transcriptional activity. Deletion of the PEA3 site within this construct substantially decreased reporter gene expression [12].
The present study was designed to compare the results from the previous in vitro promoter analysis with results generated by these in vivo electroporation studies. The differences observed between the two methods suggest that in vivo electroporation is a superior method for evaluating the promoters of genes expressed in a tissue-specific manner.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal adult male Sprague-Dawley rats (Hilltop Laboratories, Philadelphia, PA) were maintained on a 12L:12D cycle with free access to food and water in the University of Virginia vivarium. All experiments complied with the regulations set forth by the Animal Welfare Act (Public Law 91-579), the Guide for the Care and Use of Laboratory Animals (NRC, 1996) published by the Department of Health and Human Services, and the policies and procedures of the University of Virginia Institutional Animal Care and Use Committee.
Preparation of Plasmids
The cres promoter constructs were derived from the mouse promoter, which is similar in structure to the rat promoter and were previously described [11]. The GGT promoter IV constructs p135LUC, p250LUC, p530LUC, p903LUC, p1976LUC, and p(–16 to –36)135LUC were described previously [12]. The inserts from these vectors were transferred from pGL3-enhancer to pGL3-basic (both from Promega Biosciences Inc., Madison, WI) using an NheI/NarI restriction digest. The resulting plasmids were named pGL3-b135, pGL3-b250, pGL3-b530, pGL3-b903, pGL3-b1976, and pGL3-b135del, respectively. To generate GGT promoter IV-EGFP, pEGFP-C3 (BD Biosciences Clontech, Palo Alto, CA) was restriction digested with NarI and XbaI. The enhanced green fluorescent protein (EGFP) fragment was then ligated into p1976LUC that had been restricted with NarI and XbaI, releasing the luciferase sequence except for 33 bp encoding the first 11 amino acids. To generate pGL3-b681, pGL3-903 was digested with MslI and XmnI to produce a 716-bp fragment that was cloned into pGL3-basic digested with SmaI. To generate pGL3-b6500, a 
phage clone-containing genomic sequence between GGT exon V and GGT promoter IV [19], kindly provided by Dr. Y. Laperche (INSERM, Créteil, France), was digested with XbaI and BglII. A 5-kb fragment was ligated into pGL3-1976 digested with NheI and BglII. The 5-kb phage fragment overlapped with approximately 500 bp at the 5' end of pGL3-b1976; thus, pGL3-b6500 contained an additional 4.5 kb of sequence 5' to the 2 kb previously published under Genbank accession number AF218050 [12]. To generate pGL3-b135mut, pGL3-b530m1-3, and pGL3-b530c, approximately 50 ng of pGL3-b135 and pGL3-b530 were subjected to PCR-based mutagenesis using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The PCR reactions (14 cycles of 95°C for 30 sec, 55°C for 1 min, and 68°C for 11 min) were done using the following pairs of primers. Bold represents the nucleotides that were mutated with the original nucleotides above the sequence.
All plasmids were confirmed by sequencing (Biomolecular Research Facility, University of Virginia, Charlottesville, VA). All plasmids used for in vivo electroporation were purified by EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA) or CsCl density gradient ultracentrifugation.
In Vivo Electroporation
Rats were anesthetized with an intraperitoneal pentobarbital sodium injection (Nembutal; Abbott Laboratories, Chicago, IL). The scrotal contents were reached by a midline abdominal incision. Micropipettes made from R-6 glass with an outer diameter of 0.9 mm and an inner diameter of 0.6 mm (Drummond Scientific Company, Broomall, PA) were pulled on a Flaming/Brown micropipette puller (Model P-97; Sutter Instrument Company, Novato, CA). The pipettes were then sharpened on soapstone to a tip diameter of 20–30 µm. For intraluminal injections, 2–5 µl of either pEGFP-N1 (BD Biosciences Clontech) or GGT promoter IV-EGFP at 2 µg/µl were injected into the lumen of an initial segment tubule. For GGT promoter IV analysis, 15 µl of DNA were injected into zones 1a and 1b (see [20] for characterization of zones) of the initial segment interstitium underneath the capsule. For cres promoter analysis, 25µl of DNA were injected into zones 1a, 1b, and 1c of the initial segment. Following the injection, the tissue was grasped between the plates (7-mm diameter) of a pair of tweezertrodes (BTX, San Diego, CA), and 8 x 50-msec pulses of 21–24 V were delivered to the tissue using an Electro Square Porator ECM 830 (BTX). The distance between the electrodes was kept constant at 0.2 cm. The testis and epididymis were returned to the scrotum, and the body wall and skin were sutured. At the indicated times, the rats were euthanized with carbon dioxide gas, and the tissue was removed, frozen in liquid nitrogen, and stored at –80°C until use. The entire initial segment was taken from rats injected with cres promoter constructs, whereas only the proximal half of the initial segment was taken from rats injected with GGT promoter IV constructs.
To examine the different length GGT promoter IV constructs, equimolar amounts of DNA were used as follows: pGL3-b135 at 2.5 µg/µl, pGL3-b250 at 2.6 µg/µl, pGL3-b530 at 2.7 µg/µl, pGL3-b681 at 2.8 µg/ µl, pGL3-b903 at 2.9 µg/µl, and pGL3-b1976 at 3.4 µg/µl. For the experiments comparing pGL3-b530 and pGL3-b6500, 2.5 µg/µl of pGL3-b6500 and 1.2 µg/µl of pGL3-b530 were used. Linearized pUC18 was added to make the total DNA mass equivalent for each injection within an experiment. To control for electroporation efficiency, 1 ng/µl of pRL-SV40 was included with every injection. To examine the cres promoter constructs, 3 µg/µl of the different cres constructs in pGL3-basic were coinjected with 50 ng/µl of pRL-TK (electroporation efficiency control).
Fluorscence Microscopy
Following electroporation, tissues were removed at 72 h and frozen in liquid nitrogen. Five-micron frozen sections were prepared by the University of Virginia Cell Science Core, and sections were analyzed with a Zeiss 410 Laser Scanning Confocal Microscope (Carl Zeiss Optical, Inc., Thornwood, NY). Integrated black-and-white images were captured and then pseudocolored.
Dual Luciferase Assay
Assays were performed with the Dual Luciferase Assay kit from Promega according to the manufacturer's instructions with minor modifications. Previously collected and frozen tissues were ground with a mortar and pestle under liquid nitrogen. Once the liquid nitrogen had evaporated, the pellets were resuspended in Luciferase Assay Reagent II. Protein determinations were made using the Bradford method (Bio-Rad Laboratories, Hercules, CA), and 50 µg of protein in 20 µl were assayed. Measurements were taken on an FB-15 luminometer (Zylux Corporation, Maryville, TN). For GGT experiments, background values were determined on the contralateral initial segments that were uninjected and unelectroporated. Background values were then subtracted from each sample, and the ratio of firefly to renilla luciferase was calculated by dividing the firefly value of each sample with the respective renilla value. For cres experiments, background values from age-matched controls were subtracted, and then ratios were calculated as described for GGT.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To test the ability of the in vivo electroporation method to express genes in the initial segment, a plasmid-encoding EGFP under the control of the cytomegalovirus (CMV) promoter was injected intraluminally into the initial segment and electroporated. After 72 h, the initial segments were removed and analyzed with integrated confocal immunofluorescence to assess the expression of EGFP. As seen in Figure 2B, intense fluorescence was observed in the cytoplasm of the epithelial cells. This expression was observed in all tubules that had been injected with the plasmid and could be visualized as much as 1 wk after electroporation (data not shown). Next, a plasmid containing the full 1976-bp GGT promoter IV controlling EGFP expression was used. Intense staining was observed in what appeared to be a vesicular compartment (Fig. 2C). This construct contains the first 11 amino acids of firefly luciferase attached to EGFP, which may be responsible for the vesicular targeting in these cells. Minor background fluorescence can be seen in the sections in which no DNA was injected (Fig. 2A). With the intraluminal injection method, we frequently experienced tubule blockages that would result in little or no flow of fluid from the testis to the epididymis. As this defeated the purpose of analyzing these promoters by this method, we adopted an interstitial method of plasmid injection. Results from the interstitial injection of the GGT promoter IV-EGFP construct are shown in Figure 2D and reveal a similar fluorescence pattern as seen with the intraluminal injection. To avoid the complications with the intraluminal injections, the remainder of the experiments were carried out with interstitial injections.
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Analysis of Initial Segment-Specific Promoters by In Vivo Electroporation
GGT promoter IV and the cres promoter have been analyzed previously in primary initial segment epithelial cell culture and anterior pituitary cell lines, respectively [11, 12]. However, because of their dependence on LTFs for expression in the initial segment, analysis of these promoters in the context of the whole tissue was performed utilizing the in vivo electroporation method. Electroporation of the previously characterized minimal cres promoter (Fig. 1A) resulted in transcriptional activity from the parental plasmid (135 in Fig. 3A). Mutation of the 3' C/EBP site (1M) had no effect on transcriptional activity, whereas mutation of the 5' C/EBP site (2M) resulted in a 54% reduction of transcriptional activity. Mutation of both sites (DM) produced no statistically significant difference in luciferase expression compared to the 2M construct even though there may have been a significant decrease with a larger number of experiments.
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Next, equal concentrations of the 135-bp GGT promoter IV construct (pGL3-b135), the 135 construct with a deleted PEA3 site (pGL3-b135del), and the 135 construct with a mutated PEA3 site (pGL3-b135mut) were electroporated into the tissue. Whereas deletion of 20 bp containing a putative PEA3 site abrogated luciferase expression, specific mutation of the PEA3 site had no significant effect on the expression from the 135-bp GGT promoter IV construct (Fig. 3B).
Analysis of the 2-kb GGT Promoter IV by In Vivo Electroporation
Because of the unexpected results obtained with pGL3-b135, experiments were carried out with the 2-kb promoter to determine if sequences 5' to the 135 bp were responsible for GGT promoter IV activity in vivo. Equimolar concentrations of pGL3-b1976 and the 5' deletion constructs of pGL3-b1976 (Fig. 1B) were electroporated into the proximal half of the initial segment. Although each construct was able to generate some level of transcriptional activity, electroporation of the pGL3-b530 construct resulted in a significant increase in transcriptional activity compared to the other constructs (Fig. 4). Truncation of pGL3-b530 to –250 bp decreased the activity of the promoter by almost 95%, indicating the presence of a positive cis-acting regulatory element(s) between –250 and –530 bp. Additionally, when the promoter region was extended to –903 and –1976 bp, transcriptional activity decreased by 94% and 97%, respectively, suggesting the presence of a cis-acting negative regulatory element between –530 and –903 bp. To further define the region containing the cis-acting negative regulatory sequence, an additional construct, pGL3-b681, was created. When this construct was used for in vivo electroporation, expression was reduced by 79% compared to pGL3-b530, suggesting that the major repressor activity was between –530 and –681 bp (Fig. 4). This sequence (–530 to –681 bp) was then subcloned in front of the SV40 early promoter in pGL3-control to determine if it could function as a general repressor. No difference was observed between pGL3-control with and without this sequence in in vivo electroporations (data not shown).
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Analysis of pGL3-b530 Mutants
Analysis of the sequence between –250 and –530 bp reveals the presence of three consensus PEA3 DNA-binding motifs found at –452 to –447 bp, –399 to –394 bp, and –369 to –364 bp in GGT promoter IV (Fig. 1B). To determine if any of these PEA3 sites were responsible for the activity of the pGL3-b530 construct in vivo, PCR-based mutagenesis was performed to change the consensus 5'-AGGAAG-3' to 5'-AGAGAG-3' beginning with the most 5' site, yielding pGL3-b530m1, pGL3-b530m2, and pGL3-b530m3, respectively. In vivo, mutation of any one of the three PEA3 sites resulted in an 88%–90% decrease in promoter activity (Fig. 5). An additional construct, pGL3-b530c, was made by mutating the sequence 5'-AGGTAG-3' at –385 to –380 bp, which closely resembles a PEA3 DNA-binding site, to 5'-AGAGAG-3'. When this construct was used for in vivo electroporation, no significant difference compared to the wild-type pGL3-b530 construct was observed (Fig. 5).
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Cloning and Analysis of Additional GGT Promoter IV Sequence
The results shown in Figure 3 suggested that pGL3-b1976 does not represent the entire GGT promoter IV. To examine more 5' sequences, a 4.5-kb fragment from a phage clone-containing rat genomic DNA between exon V and promoter IV [19] was cloned into pGL3-b1976. In vivo electroporations were carried out with this construct, pGL3-b6500, and compared to electroporations done with equimolar concentrations of pGL3-b530. As shown in Figure 6, pGL3-b6500 expression was only 7% of pGL3-b530. These results were similar to pGL3-b1976, which had only 3% the expression of pGL3-b530 (Fig. 5), suggesting that additional 5' sequences may be involved in the control of GGT promoter IV.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of the cres promoter had been carried out previously in LTß2 gonadotroph cells, which normally express cres [11]. The transcriptional activity of the 135-bp minimal promoter was dependent on two C/EBP DNA-binding sites found within this sequence. The same promoter was used in this study. Although the promoter is derived from the mouse sequence, the rat promoter is similar to the mouse, and the expression of cres is restricted to the rat initial segment as it is in the mouse (unpublished observations). In this study, only the 5' C/EBP element in the cres promoter was necessary for promoter activity in vivo in the initial segment, suggesting that only the 5' site may be necessary for the activity of this promoter in vivo (Fig. 3A). In addition, there may be differences in the transcriptional control of the cres promoter in the anterior pituitary compared to the initial segment that are revealed by these in vivo studies.
Substantial differences were observed when GGT promoter IV was analyzed in vivo. In primary cell cultures of initial segment epithelial cells, cotransfection of PEA3 with the various GGT promoter IV constructs was able to activate a promoter truncated at –135 bp [12]. In vivo, the 135-bp construct had some transcriptional activity, which was equivalent to expression from the longer pGL3-b250 construct. However, the activity of both pGL3-b135 and pGL3-b250 was approximately 10% of that observed when the promoter was extended to –530 bp (Fig. 4), indicating the presence of a site or sites between –250 and –530 bp that result in increased transcriptional activity. Three consensus PEA3 sites are found within this region (Fig. 1B). When the core GGAA was mutated within any one of these sites, promoter activity decreased to approximately 10% of that seen with pGL3-b530, suggesting that all three of these sites are necessary for the increased activity seen with pGL3-b530 (Fig. 5). A control mutation made in a similar site within this region had no effect on promoter activity. These studies are in agreement with previous data from electrophoretic mobility shift assays that showed a protein from initial segment nuclear extracts bound an oligonucleotide containing the PEA3 site at –399 to –394 bp [21]. Binding of this protein was blocked when a PEA3 monoclonal antibody was added to the extracts [21]. Of particular interest is the fact that all three PEA3 sites appear to be necessary for pGL3-b530LUC activity, suggesting a coordinated regulation of this construct through these sites. This pattern of PEA3 DNA-binding sites appears to be unique to GGT promoter IV. PEA3 DNA-binding sites have been demonstrated to play roles in the regulation of urokinase plasminogen activator, matrix metalloproteinase 1, and gelatinase B promoters [22–24]. However, in these promoters, PEA3 acts in conjunction with adjacent AP-1 sites. Interestingly, there is an AP-1 site in the b135 construct, which, when mutated, has no effect on transcriptional activity (data not shown). Future studies are aimed at identifying which of the PEA3 family members binds to these sites and the stoichiometry of binding. Variable binding to these sites may play a role in the regulation of the level of GGT mRNA IV expression.
When GGT promoter IV was extended to –681 and –903 bp, a 79% and 95% decrease in promoter activity was observed, respectively, suggesting the presence of one or more negative cis-regulatory elements within this region (Fig. 4). However, when the sequence between –530 and –681 bp was cloned in front of the SV40 promoter of pGL3-control, no change in transcriptional activity of the SV40 promoter was observed (data not shown), suggesting that this sequence does not act as a general repressor. The action of this repressor sequence may be context dependent. For example, the PEA3 sites between –250 and –530 bp may be necessary for repressor activity. Analysis of the sequence between –530 and –903 bp reveals a highly AT-rich region. Some homeodomain proteins, which bind to AT-rich sequences, act as repressors of transcription. The homeodomain protein, Nkx3.1, represses the activity of the prostate-derived Ets factor (PDEF), another Ets family member, on the prostate-specific antigen promoter in prostate cells [25]. Several other potential transcription factor binding sites are found within this region, including two GATA-1 sites. GATA-1 can interact with Ets family member, PU.1, and interfere with its ability to transactivate myeloid target genes [26]. Alternatively, these experiments raise the possibility that perhaps structural hindrance from the increasing size of the promoter rather than a repressor may be the cause of the decreased transcriptional activity. This idea is reinforced by the results obtained with pGL3-b6500 (Fig. 6) and raises the question about what happens to this region in vivo when this sequence is found in the context of chromosomal DNA, which is affected by the regulation of chromatin condensation and unwinding. Future studies are aimed at elucidating the mechanism for the decreased transcriptional activity observed with the sequences upstream of –530 bp.
When the 2-kb promoter sequence was used to assay for promoter activity in the in vivo electroporations, minimal expression was observed compared to pGL3-b530, which suggested that perhaps the entire promoter sequence had not been identified (Fig. 4). In an attempt to analyze more 5' promoter sequence, an additional construct was made that contained 4.5 kb of sequence 5' to the 2 kb previously cloned [12]. When this construct was used for in vivo electroporations, no additional promoter activity was observed (Fig. 6). One explanation for these results may be that pGL3-b6500 does not represent the entire promoter sequence. Approximately 10 kb of genomic DNA sequence exist between GGT 5' UTR exon V and the transcriptional start site for GGT mRNA IV. These experiments examined the effects of only 6.5 kb of this sequence. Another explanation for these results may be the existence of additional cis-acting regulatory elements upstream of the GGT gene locus, such as an enhancer or locus control region (LCR). These elements would not be identified by these in vivo electroporation studies. LCRs are functionally characterized as DNA sequences that allow for copy number-dependent expression of transgenes in mice regardless of the chromosomal integration site [27]. LCRs have been described for groups of genes that are coordinately regulated, such as the ß-globin gene locus [27], the human growth hormone (hGH) locus [28], and the red and green cone pigment gene locus [29]. Indeed, analysis of the other GGT promoters revealed similar results to those depicted in Figure 3 [19, 30–33], suggesting that additional sequences may be necessary for correct expression from the GGT locus. Possibly, these upstream sequences control the general expression from the GGT locus, whereas cell type-specific regulation comes from cis-regulatory elements present within the promoters themselves. Finally, structural hindrance from the increased sequence length as mentioned in the previous paragraph may also be contributing to the decreased expression seen with pGL3-b6500.
One of the most exciting benefits of the in vivo electroporation method for studying initial segment gene expression is the ability to assess the effects of LTFs on initial segment promoter activity by comparing results from experiments done either in the presence or absence of LTFs (±EDL). This type of experiment would allow one to determine if there were additional sites in the promoter sequence that are sensitive to the effects of LTFs. Studies are currently being performed to address these issues.
In conclusion, this report highlights the benefits of using in vivo electroporation as a method to analyze promoters in the rodent epididymis, in particular the initial segment, which is highly dependent on LTFs for the expression of multiple genes. The results of the experiments presented here demonstrate substantial differences compared with results obtained by in vitro cell culture methods [11, 12], emphasizing the importance of evaluating these genes in their native environment. However, this method could be adapted for use in any tissue that can be easily manipulated, and it offers the advantage of testing promoters in the intact tissue rather than cell culture.
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ACKNOWLEDGMENTS |
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phage clone-containing genomic GGT sequence. |
FOOTNOTES |
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2 Correspondence: Barry T. Hinton, P.O. Box 800732, University of Virginia Health System, Charlottesville, VA 22908. FAX: 434 982 3912; bth7c{at}virginia.edu
Received: 21 November 2003.
First decision: 8 December 2003.
Accepted: 9 April 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glutamyl transpeptidase messenger ribonucleic acid transcripts in the adult rat epididymis is differentially regulated by androgens and testicular factors in a region-specific manner. Endocrinology 1994 135:1146-1156[Abstract]-glutamyl transpeptidase gene expression in undifferentiated rat transformed liver cells. Biochem J 1997 326:pt 2311-320-glutamyl transpeptidase promoter that is expressed and regulated in the liver and hepatoma cells. J Biol Chem 1994 269:14878-14884-glutamyl transpeptidase gene: structure of a promoter active in the kidney. Biochemistry 1991 30:1618-1623[CrossRef][Medline]-glutamyl transpeptidase in rat kidney and epididymis. Biochemistry 1992 31:9190-9196[CrossRef][Medline]This article has been cited by other articles:
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  W. R. Lagor, R. Heller, E. D. De Groh, and G. C. Ness Functional Analysis of the Hepatic HMG-CoA Reductase Promoter by In Vivo Electroporation Experimental Biology and Medicine, March 1, 2007; 232(3): 353 - 361. [Abstract] [Full Text] [PDF]
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 L. Yang, S. A. Fox, J. L. Kirby, B. V. Troan, and B. T. Hinton Putative Regulation of Expression of Members of the Ets Variant 4 Transcription Factor Family and Their Downstream Targets in the Rat Epididymis Biol Reprod, April 1, 2006; 74(4): 714 - 720. [Abstract] [Full Text] [PDF]
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 E. V. Zubkova and B. Robaire Editorial Commentary J Androl, September 1, 2005; 26(5): 638 - 640. [Full Text] [PDF]
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