Androgen Responsiveness of Mouse Kidney ß-Glucuronidase Requires 5'-Flanking and Intragenic Gus-s Sequences

S. Thornton, D. W. Thomas, P. M. Gallagher and R. E. Ganschow

Graduate Program in Developmental Biology (S.T., D.W.T.) College of Medicine University of Cincinnati Cincinnati, Ohio 45221
Children’s Hospital Research Foundation (P.M.G., R.E.G.) Cincinnati, Ohio 45229


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetics studies of natural variants of the androgen response of mouse ß-glucuronidase (GUS) reveal a cis-active element closely linked to the GUS structural gene (Gus-s) that is necessary for this kidney-specific response. Results of our previous studies suggested sequences within or near an androgen-inducible deoxyribonuclease I-hypersensitive site (DH site) located in the ninth intron of Gus-s are associated with the androgen response of GUS. Using transgenic mice, we now demonstrate that at least two regions of sequence within Gus-s are involved in regulating the androgen response of GUS. The first, located within 3.8 kb of Gus-s 5'-flanking sequence, directs the response and its tissue specificity, while the second, located within a 6.4-kb fragment of Gus-s extending from the third through the ninth intron of Gus-s, protects the androgen responsiveness of the transgene from repressive influences of the insertion site.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The transcriptional regulation of gene expression by steroid hormones is primarily mediated by steroid receptors (reviewed in Refs. 1–3). Upon hormone binding, these receptors are able to interact with hormone-response elements (HREs) associated with hormone-responsive genes. The receptors for androgens, as well as those for glucocorticoids, progesterone, and mineralocorticoids, all recognize the same consensus HRE, raising the unresolved issue of how individual hormone receptors discriminate among potential HREs.

Several potential HREs have been identified in androgen-regulated genes, including the rat 20-kDa protein gene, the rat C3(1) gene of the prostatic binding protein, the mouse sex-limited protein gene, the human prostatic-specific kallikrein gene, and the rat probasin gene (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). In vitro analysis of these elements indicates sequences in addition to the HRE are necessary to obtain a maximal androgen response, implying that a specific combination of factors that bind these sequences may be necessary to obtain hormone responsiveness.

Functional analysis of androgen-response elements has not been performed extensively in vivo. In a transgenic mouse study of rat C3(1) gene expression, three of four transgenic lines selectively expressed the transgene in the ventral prostate (14). The expression of this transgene was stimulated by androgen; however, this expression was neither copy number-dependent nor position-independent (14).

Transgenic mouse studies utilizing putative androgen-response elements of the androgen-regulated probasin gene promoter showed that three of five transgenic lines were able to exhibit the hormonally and developmentally correct pattern of probasin gene expression. When a matrix attachment region (MAR) element was cointegrated with the probasin transgene sequences, three of three transgenic lines mimicked the hormonal and developmental response of probasin to androgen but still lacked copy number-dependent expression (15). This result suggests that consistent androgen regulation of transgenes may require elements similar to MARs to overcome chromatin repression of hormone induction. Furthermore, additional undefined elements are also required, since copy number dependence is not achieved.

It is well established that chromatin structure plays a role in steroid hormone gene regulation (1, 3, 16, 17). Glucocorticoid induction of the mouse mammary tumor virus (MMTV) promoter is associated with alterations in chromatin structure that allow the acquisition of transcription factors necessary for the hormonal response (18, 19). Chromatin structure may also be involved in the steroid specificity of the MMTV response. Studies of the MMTV promoter reveal that glucocorticoid receptors can activate either transient or stable transfected templates, whereas progesterone receptors can only activate transiently transfected templates (16). Furthermore, the failure of progesterone to induce transcription in stably transfected templates correlates with the failure to induce chromatin structural alterations (16). These results imply that the effects of steroid receptors on the transcription of a DNA template may differ depending upon whether the DNA is transiently or stably transfected into a cell. Thus, appropriate assessment of endogenous transcriptional responses to hormones may require an organized chromatin environment as is provided by transgenic analysis.

We are utilizing the kidney-specific androgen response of mouse ß-glucuronidase (GUS) as a model of hormonal control of mammalian gene regulation. Androgens induce hypertrophy of mouse proximal tubule and Bowman’s capsule cells as well as RNA accumulation and protein synthesis of specific gene products, including GUS (20).

Natural variants of the androgen response of GUS among strains of laboratory mice have provided a convenient and powerful system for analysis of this response. Our attention has been focused on three of these variants, which are classified as follows: A haplotype strains, which exhibit a strong response to androgen; B haplotype strains, which exhibit a low response; and the OR haplotype strain, which exhibits no response (21, 22). Genetic studies suggest these differences are under the control of three alleles of a cis-active element(s) that is tightly linked to the GUS structural gene (22, 23). Further lines of evidence suggest that sequences closely associated with an androgen-inducible deoxyribonuclease I (DNase I)-hypersensitive site (DH site) within the ninth intron of Gus-s are important for the androgen regulation of GUS (24). First, this DH site is present in chromatin of androgen-responsive GUS haplotypes but absent in chromatin of the nonresponsive OR haplotype. Second, a consensus HRE is present in the ninth intron sequence of androgen-responsive GUS haplotypes but is deleted in the nonresponsive haplotype. Third, a kidney-specific, androgen-inducible factor binds to sequences within this androgen-inducible DH site (24).

In the present study, we utilize transgenic mice to functionally assess Gus-s sequences for their ability to direct the androgen responsiveness of a luciferase (LUC) reporter gene. Results of these studies demonstrate that Gus-s 5'-flanking sequences are necessary and sufficient for the kidney-specific, androgen regulation of the transgene. In addition, sequences within a 6.4-kb region extending from the third through the ninth intron of Gus-s protect the androgen responsiveness of the transgene from repressive influences of the insertion site.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gus-s 5'-Flanking Sequences and Not Ninth Intron Sequences Direct Androgen Responsiveness of Luciferase Reporter Expression
Several lines of circumstantial evidence, summarized in the Introduction, suggest that elements controlling the kidney-specific, androgen response of GUS lie within the ninth intron of Gus-s (24). To test whether sequences within the Gus-s ninth intron can direct the kidney-specific, androgen response of a reporter gene in transgenic mice, we created two transgenic constructs designated GusLuc1.8 and GusLuc. Both contain a luciferase reporter gene driven by 3.8 kb of 5'-flanking Gus-s sequence. GusLuc1.8 contains an additional 1.8-kb region of Gus-s, encompassing the androgen-inducible DNase I-hypersensitive site of the ninth intron, downstream of the LUC reporter gene (see Fig. 1Go).



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Figure 1. Transgenic Constructs

Figure indicates sequences contained within the three transgenes; diagonal stripes represent Gus-s 5'-kb flanking region; dots on white background indicate luciferase cDNA; the Gus-s endogenous gene is shown in a series of open and filled boxes, with open boxes representing introns and filled boxes representing exons. The 1.8- and 6.4-kb downstream Gus-s fragments are indicated by lines below Gus-s. Approximate locations of DNase I- hypersensitive sites 3 and 4 are indicated above Gus-s. (Generation of transgenic constructs is explained in Materials and Methods.)

 
Comparison of kidney LUC activity levels among the GusLuc1.8 transgenic lines shows that one line (line 54) exhibits a response of LUC to androgen, suggesting that sequences necessary for the GUS response are present in this construct (see Fig. 2AGo). While basal levels of kidney LUC activity are detectable in seven of nine lines, two transgenic lines exhibit detectable levels of LUC activity only in spleen (data not shown). Of the lines that show basal levels of kidney LUC activity, assessment of one transgenic line consisted of only one available male, which showed low levels of LUC activity in kidney, liver, and spleen (data not shown). Thus, LUC expression is observed in all GusLuc1.8 lines tested, but only line 54 shows a response of LUC to androgen in the kidney.



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Figure 2. Androgen Response of Kidney Luciferase Activity

A, Kidney extracts from untreated and 7-day-treated GusLuc1.8 females were assayed for LUC activity as described in Materials and Methods. B, Kidney extracts from untreated and 14-day treated GusLuc females were assayed for LUC activity as described in Materials and Methods. Bars represent the mean value of at least five mice, with error bars representing ±SEM.

 
Since two separate regions of Gus-s are represented in the GusLuc1.8 transgene, we also asked whether 5'-flanking sequences alone are sufficient for the observed response. To address this question we used GusLuc, which essentially is GusLuc1.8 devoid of the Gus-s ninth intron sequences (see Fig. 1Go). Thirteen transgenic lines were generated from GusLuc, and each was tested for the ability of the transgenic LUC gene and the endogenous GUS gene to respond to androgen.

Analysis of kidney extracts from untreated and 14-day treated GusLuc transgenic mice reveals that while all 13 lines exhibit basal levels of LUC, four lines show an increase in LUC activity upon androgen treatment (See Fig. 2BGo), demonstrating that 5'-flanking sequences alone are sufficient for mediating the androgen response of GUS. One GusLuc line (line 4) shows a strong response of kidney LUC, three lines (lines 1, 25, and 27) show a weak response (see Fig. 2BGo), and nine lines show no response. Of the responding lines, only line 4 exhibited a response of LUC activity that was both highly significant (P < 0.05) and kidney-specific (similar to endogenous GUS activity).

Response Characteristics of the GusLuc1.8 and GusLuc Reporter Genes Are Similar to Those of Endogenous GUS
To determine whether the Gus-s sequences controlling the androgen response of GusLuc1.8 line 54 and GusLuc line 4 can direct a response characteristic of the endogenous GUS response, both the tissue specificity and the kinetics of the transgene responses were examined.

Since the androgen response of GUS is kidney-specific, we tested whether Gus-s sequences present in the transgene of GusLuc1.8 line 54 and GusLuc line 4 mice are able to direct a tissue-specific response. Comparison of LUC and GUS activity levels between untreated and treated females of GusLuc1.8 line 54 show increased LUC and GUS activity levels in kidney but not in liver or spleen (see Fig. 3AGo). Although exhibiting a low basal level of LUC expression, other tissues, including heart, lung, ovary, uterus, large intestine, small intestine, skin, skeletal muscle, stomach and brain, show no response of LUC to androgen (our unpublished data and Ref.25). Similarly, comparison of LUC and GUS activity levels between treated and untreated females of GusLuc line 4 show increased LUC and GUS activity levels in kidney but not in liver or spleen (Fig. 3BGo). These results demonstrate that Gus-s 5'-flanking sequences within the transgenes are capable of directing the kidney-specific, androgen response of GUS.



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Figure 3. Tissue Specificity of GUS and LUC Activities in GusLuc1.8 Line 54 (A) and GusLuc Line 4 Mice (B)

Kidney, liver, and spleen extracts from untreated and 14-day-treated females were assayed for GUS and LUC activity. Fold induction is the mean value (n >= 5) of tissue extracts from 14-day treated mice divided by the mean value (n >= 5) of the tissue extracts from untreated mice.

 
The androgen response of GUS in mouse kidney is characterized by a 1- or 2-day lag, followed by an unusually slow rise of GUS that reaches peak levels at approximately 21 days after initiation of treatment. Other androgen responses observed in the mouse kidney are more rapid than GUS, usually reaching their peak levels after 2–3 days of androgen treatment (26). Since the temporal expression pattern of the GUS response is distinctive, the temporal response of LUC to androgen was examined in GusLuc1.8 line 54 and GusLuc line 4 mice. Figure 4Go shows LUC and GUS kidney activity levels of these two transgenic mouse lines at time points from 0–21 days of androgen treatment. Since both activities show similar kinetic responses, we infer that elements common to both transgenes control not only the tissue specificity of the GUS response but also its unique temporal expression pattern.



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Figure 4. Kinetics of the Androgen Response of LUC and GUS in GusLuc1.8 Line 54 (A) and GusLuc Line 4 Mice (B)

Females were treated with androgen and killed on the days indicated. LUC and GUS values were obtained from assays of kidney extracts as descibed in Materials and Methods. Plotted values are the mean of at least five mice.

 
Inclusion of Additional Gus-s Sequences Ensures Position Site-Independent Transgene Responsiveness
The low frequency of responsiveness of kidney LUC among the GusLuc and GusLuc1.8 transgenic lines suggests that factors independent of transgene sequences have a negative influence upon transgene expression. Since chromatin has been shown to exert such effects, it is possible that the nonresponsive transgenes had inserted into chromosomal regions that repress the androgen response, whereas the responsive transgenes had inserted into chromosomal regions that do not repress the androgen response. To determine whether the presence of additional Gus-s elements may overcome the observed repression of the response of the transgene, we created a construct designated GusLucA6.4F, which includes 6.4 kb of Gus-s sequence containing two DNase I-hypersensitive sites, which are potential sites for cis-acting regulatory elements (see Fig. 1Go). This construct, as compared with GusLuc1.8, contains an additional constituitive DNase I-hypersensitive site located in intron 4, along with the androgen-inducible site in intron 9 (24).

Comparison of kidney LUC activity levels between untreated and treated GusLucA6.4F transgenic mice shows that each of six lines examined exhibit an androgen response of the LUC reporter (see Fig. 5Go). In five of these six lines the response is highly significant (P < 0.05). While at early time points androgen-treated animals of line 21 showed a higher level of LUC expression, statistically significant differences (P < 0.05) were not observed until 21 days after treatment (data not shown).



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Figure 5. Androgen Response of Kidney Luciferase Activity in GusLucA6.4F Mice

Kidney extracts from untreated and 14-day treated GusLucA6.4F females were assayed for LUC activity as described in Materials and Methods. Bars represent the mean value of at least five mice, with error bars representing ± SEM. Note: values are plotted on a logarithmic broken axis. The androgen-treated mean ± SEM values for lines 24, 27, and 53 are, respectively, 481.4 ± 116.8, 174.5 ± 51.9, and 252.5 ± 37.4.

 
In contrast to the previously tested transgenic constructs, this high frequency of androgen responsiveness of GusLucA6.4F expression suggests that sequences within this 6.4-kb Gus-s fragment function to protect the androgen responsiveness of the transgene. However, the extent of the androgen response in each line is variable, and neither basal nor induced LUC activity levels are copy number-dependent (Table 1Go), suggesting that the protection from external influences is not complete. Nevertheless, since each of the six transgenic lines show an androgen response, the presence of the Gus-s 6.4-kb fragment now ensures the position site independence of the transgenic response.


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Table 1. Androgen Responsiveness of the GusLucA6.4F Transgene

 
Response Characteristics of the GusLucA6.4F Reporter Gene Are Similar to Those of Endogenous GUS
To determine whether Gus-s sequences present in the GusLucA6.4F transgene direct a "GUS-like" response, the tissue specificity and kinetics of the reporter transgene response were examined.

LUC and GUS values for kidney, spleen, and liver were compared between untreated and treated GusLucA6.4F mice. In all lines, an androgen response of LUC and GUS in kidney is observed (see Fig. 6Go). Spleen extracts exhibit no significant increase in LUC or GUS activity after 14 days of androgen treatment. However, three of the six GusLucA6.4F lines (lines 16, 24, and 51) show increases in liver LUC activity, which are substantially lower than the increases in LUC activity observed in kidney after androgen treatment (see Fig. 6Go). This increase is significant (P < 0.05) only in line 51. GUS activity also increases somewhat in the liver, suggesting that the androgen induction of GUS is "leaky" in liver. This leakiness may be amplified in the context of the transgene. Nevertheless, the tissue distribution of the androgen responsiveness of the luciferase reporter is similar to that of endogenous GUS.



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Figure 6. Tissue Specificity of GusLucA6.4F GUS and LUC Activities

Kidney, liver, and spleen extracts from untreated and 14-day treated GusLucA6.4F females were assayed for LUC (panel A) and GUS (panel B) activities. Fold induction is the mean value (n >= 5) of 14-day treated mice divided by the mean value (n >= 5) of tissue extracts from untreated mice.

 
To determine whether sequences present in the GusLucA6.4F transgene can confer a temporal response pattern similar to that of the endogenous GUS response, three GusLucA6.4F transgenic lines were examined separately for the kinetics of both the transgenic LUC and the endogenous GUS responses. As shown in Fig. 7Go, results of treatment of females from GusLucA6.4F lines 24, 27, and 51 show that all three GusLucA6.4F transgenic lines, like the responsive GusLuc and GusLuc1.8 transgenic lines, exhibit a LUC transgenic response that parallels that of the endogenous GUS response.



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Figure 7. Kinetics of the Androgen Response of LUC and GUS Activites in GusLucA6.4F Mice

Female mice from ines 24, 27, and 51 (panels A, B, and C, respectively) were treated with androgen and killed on the days indicated. LUC and GUS values were obtained from assays of kidney extracts as described in Materials and Methods. Plotted values are the mean of at least five mice, except for 7-day values for lines 27 and 51, which are the mean of two mice.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our results demonstrate that at least two distinct DNA elements regulate the kidney-specific androgen response of GUS in mice. One of these, located within the proximal 3.8 kb of 5'-flanking sequence of Gus-s, is sufficient to direct a GUS-like response of a reporter gene in transgenic mice, while the other, located within 6.4 kb of Gus-s intragenic sequence, appears to protect the androgen responsiveness from insertion site repression.

Our finding implies the presence of factors that interact with an element or elements within the 5'-flanking region of Gus-s. Such factors could be acting with 5'-flanking sequence in a variety of ways. Hormone-inducible responses are generally mediated through the hormone receptor and cis-active HREs (3, 20). The mechanism of steroid hormone receptor interaction with the transcriptional machinery is unknown, but studies suggest hormone receptors can interact directly or indirectly with general or sequence-specific transcription factors (reviewed in Refs. 1, 3, 27, and 28).

In the context of the transgenic constructs used in our studies, the Gus-s 5'-flanking region by itself supports a low frequency of androgen responsiveness, indicating that the transgene integration site greatly influences the ability of the transgene to respond. However, we believe that the tissue-specific, androgen response of the transgenic LUC reporter gene in GusLuc line 4, and in GusLuc1.8 line 54 are directed by sequences contained within these transgenes and not by the transgene integration site. First of all, it is unlikely that of the 22 GusLuc and GusLuc1.8 transgenic lines tested, two lines are randomly inserted into a similar transgene integration site that is able to confer a GUS-like transgene response. Second, because the temporal expression pattern of the androgen response of GUS is unique (21, 26), it is unlikely that another gene locus could confer a similar hormonal response. Furthermore, since endogenous GUS levels are normal in all transgenic lines tested, and Southern analysis of transgenic mouse lines indicates the presence of an intact Gus-s gene (data not shown), we assume that the transgenes have not disrupted the Gus-s locus. Thus, we infer that Gus-s 5'-flanking sequences, and not the insertion site, direct the kidney-specific, androgen response of the LUC reporter gene in responsive transgenic lines.

Basal levels of LUC are observed in all transgenic lines, indicating that the minimal elements needed for basal expression are present in the GusLuc construct. Furthermore, basal LUC levels vary widely among the transgenic lines, and this variation is not a function of transgene copy number (data not shown), implying that the insertion site is affecting basal levels of expression. However, the low frequency of androgen responsiveness of luciferase in mice transgenic for GusLuc and GusLuc1.8 suggests that a general repression of the hormonal response of the transgene is occurring in most lines. Since it is likely that this behavior is a function of the structure of the chromatin into which the transgene inserts, we hypothesize that responsive lines insert into a region of chromatin that allows factors necessary for the hormonal response to interact through elements within the 5'-flanking region, while nonresponsive lines insert into a chromatin region in which such factors cannot access their respective elements. The observation that androgen responsiveness occurs in kidney when the 6.4-kb Gus-s intragenic fragment is included in the transgene suggests that these additional sequences establish a chromatin configuration favorable for androgen responsiveness.

Elements crucial to appropriate genomic organization and correctly regulated gene expression have been identified in a number of laboratories. A locus control region was first described for the ß-globin locus utilizing a human ß-globin transgene in mice (29). This transgene exhibited tissue-specific expression at a level directly related to its copy number and independent of its genomic transgene integration site (29). Studies of MARs or scaffold attachment regions found in several genes from various species indicate these regions may insulate genes as a single transcription unit (30). These elements are typically AT-rich (70%) and have been shown biochemically to be associated with the nuclear matrix (30, 31, 32). Stable transfections of both chicken and heterologous cells with a reporter gene flanked by the 5'-chicken lysozyme MAR indicate that the MAR sequences allow for elevated and position-independent gene activity (30, 33).

Our results are similar to those from transgenic mouse studies in which MAR sequences regulate, in a position-independent manner, the hormonal and developmental expression of the whey-acidic-protein transgene (34) and a transgene containing the androgen-response elements of the rat probasin gene (15). Expression of these transgenes, when cointegrated with MAR sequences, increased from six of 12 lines to 11 of 11 lines for the rat whey acidic protein transgene, and from three of five lines to three of three lines for the probasin gene promoter-driven transgene. However, in both instances the transgene expression is not copy number-dependent, indicating that sequences are not present for this function. These functional studies of MAR sequences, as well as the present studies, suggest that elements influencing the higher order chromatin structure of the DNA have an effect on the hormonal responsiveness of the transgene but may not be able to completely insulate the transgene from all insertion site effects.

Our results do not rule out the possibility that the spatial orientation of the ninth intron, relative to the Gus-s 5'-flanking region, is critical for protecting the ability of the transgene to respond to androgen, since the distance from the 5'-flanking sequence to the ninth intron sequence within the GusLucA6.4F transgene is similar to that within the endogenous gene (see Fig. 1Go). If such spacing is critical, then it is possible that, in contrast to GusLucA6.4F, the failure of GusLuc1.8 to provide protection of the response is a function of the reduced distance between the Gus-s ninth intron sequences and the Gus-s 5'-flanking sequences in this construct.

Further functional analysis of Gus-s sequences utilizing the GusLucA6.4F construct in transgenic mice will allow identification of specific sequences responsible for the androgen responsiveness of GUS and may define sequences that protect hormonal response elements from repressive influences of the chromatin environment.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transgenic Constructs
pGusLuc was derived from {lambda}AGus-4, a {lambda}-phage clone containing the entire Gus-s coding region plus approximately 4 kb upstream and 1.5 kb downstream (35), and from pXP2, a plasmid containing a luciferase expression vector (American Type Culture Collection, Rockville, MD). To generate pGusLuc, 3.8 kb of the Gus-s promoter region were inserted upstream of the luciferase gene in pXP2 as follows.

A 581-bp PCR product, encompassing the area from nucleotides +11 to -570 of Gus-s, was inserted into pXP2 upstream of the luciferase gene; subsequently, a 3.7-kb HindIII/XhoI fragment of the 5'-region of Gus-s was cloned into the pXP2 polylinker HindIII site and an XhoI site located at position -90 of the promoter PCR product. The entire pXP2 insert, including the 3.8-kb Gus-s promoter region and luciferase cDNA, was excised and cloned into Bluescript SK II- (Stratagene, La Jolla, CA), resulting in pGusLuc.

pGusLuc1.8 was created by cloning a 1.8-kb BamHI restriction fragment (which includes base pairs 10,075–11,920 of Gus-s) into pGusLuc via a BamHI site located downstream of the luciferase cDNA (see Fig. 1Go). pGusLucA6.4F was created by cloning a 6391-bp ApaLI restriction fragment, blunt-ended with Klenow (which includes base pairs 4946–11,336 of Gus-s) in the forward orientation into the pGusLuc SmaI site located downstream of the luciferase cDNA (see Fig. 1Go).

To obtain DNA for injection into mouse embryos, plasmid DNAs were cut with appropriate restriction enzymes, and DNA fragments were isolated by gel electrophoresis and subsequent purification from low melt agarose using either the Prep-a-Gene DNA purification kit (Bio-Rad, Hercules, CA) or phenol/chloroform extractions and ethanol precipitation of the DNA. Any further contaminants were removed by applying DNAs to an elutip column (Schleicher & Schuell, Keene, NH) and then precipitating the eluate with ethanol. DNAs were injected into embryos by one of the authors (D.W.T.) and by the Children’s Hospital Medical Center transgenic core facility. Founder mice strains were B6/C3H, which are B haplotype for the Gus-r allele (36).

Animal Procedures
For androgen treatment of mice, animals 2–4 months of age were anesthetized by intraperitoneal injection of 0.015–0.017 ml 2.5% (vol/vol) avertin/g body weight (37). A 25-mg pellet of testosterone was then inserted subcutaneously at the nape of the neck after the animals were anesthetized. At the appropriate time intervals thereafter, mice were euthanized by CO2 inhalation and cervical dislocation.

All mouse strains used were obtained from either Jackson Laboratories (Bar Harbor, ME) or Charles River Laboratories, Inc. (Wilmington, MA). All animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Children’s Hospital Research Foundation Institutional Animal Care and Use Committee under animal use protocol numbers 2D02015 and 5D02016.

Tissue Extract Preparation
Tissues were placed in 400 µl of ddH2O and homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury NY). To obtain tissue extracts, homogenates were incubated in 1x Reporter Lysis Buffer (Promega Corp., Madison, WI) at room temperature for 20 min. Cellular debris was pelleted by centrifugation at 12,000 x g, for 5 min at 4 C in a microcentrifuge. The supernatants were placed on ice during use and subsequently stored at -70 C.

Luciferase Assays
Luciferase assay reagent (Promega) was resuspended and loaded into a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). Ten-microliter aliquots of tissue extracts were assayed in duplicate for 10 sec. Background luciferase activity was measured using either empty tubes or 10 µl of 1x lysis buffer containing either 10 mg/ml BSA or nontransgenic mouse tissue extracts, each of which gave similar values. Background values were subtracted from the mean of the duplicate readings to obtain relative light units. Protein concentrations were measured by the bicinchoninic acid assay system (Pierce, Rockford, IL) using BSA as standard. Luciferase activity is expressed as relative light units/µg protein.

Glucuronidase Assays
GUS activity was determined using p-nitrophenyl-ß-D-glucuronide as substrate at 56 C (38). GUS activity is expressed as activity units /mg protein, where 1 activity unit equals the amount of enzyme necessary to liberate 1 µmol p-nitrophenol/h at 56 C. Protein concentrations were measured by the bicinchoninic acid assay system (Pierce) using BSA as standard.

Statistical Analysis
The significance of differences between the mean values of two sample populations was determined (P <= 0.05, unless otherwise stated) by rejection or acceptance of the null hypothesis that states these mean values are equal (39). When the null hypothesis is true, the test statistic follows Student’s t distribution with n1 + n2 - 2 degrees of freedom.


    ACKNOWLEDGMENTS
 
We thank Dr. Dan Wiginton and Dr. Jun Ma for critical reading of the manuscript, Scott Hartman and Kim Kurak for technical assistance, and Bonnie Sprecher for assistance with statistical analyses.


    FOOTNOTES
 
Address requests for reprints to: Roger E. Ganschow, Ph.D., Children’s Hospital Research Foundation, Division of Developmental Biology, 3333 Burnet Avenue, Cincinnati, Ohio 45229.

This work was supported by NIH Grants DK-14770 and HD-07463.

Received for publication October 2, 1997. Revision received December 9, 1997. Accepted for publication December 11, 1997.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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