TECHNOLOGY REPORT
Human CYP11A1 Promoter Drives Cre Recombinase
Expression in the Brain in Addition to Adrenals
and Gonads
Hsu-Shui Wu,1,yHui-Ting Lin,1,yChi-Kuang Leo Wang,2Yen-Feng Chiang,1 Hsueh-Ping Chu,2and Meng-Chun Hu1,*
1
Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China
2
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China
Received 17 October 2006; Accepted 29 November 2006
Summary: The first step of steroid biosynthesis is cata-lyzed by cytochrome P450scc, encoded byCYP11A1. To achieve steroidogenic tissue-specific inactivation of genes in vivo by the Cre-loxP approach, we used the 4.4-kb regulatory region of the humanCYP11A1 gene to drive Cre recombinase expression in the tissues that produce steroids. The resulting SCC-Cre mice express high levels of Cre in the adrenal cortex and gonads at the same sites as that for the endogenous CYP11A1 expression. In addition, Cre activity was found in the diencephalon and midbrain. In the developing brain, the Cre activity was first detected in the embryonic day 10.5. Our study is the first to show that the 4.4-kbCYP11A1 promoter is transcriptionally active in the brain in vivo. genesis 45:59–65, 2007. VVC2007 Wiley-Liss, Inc.
Key words: CYP11A1; Cre; adrenal; gonad; diencephalon; midbrain; hippocampus; VMHDM
Conventional gene targeting that introduces permanent genetic changes into the germ line of mice by homolo-gous recombination has been widely employed to inves-tigate gene functions. The complete loss of genes that are essential for embryogenesis may result in the pheno-type of embryonic lethality. Embryonic lethality prevents the analysis of gene functions at the later developmental stages or in adult tissues. The disruption of genes in all cells and tissues also precludes the study of gene func-tions in a specific cell type or at a given time. To over-come the limitations, site-specific recombination pro-vides a useful approach to achieve conditional and tis-sue-specific gene inactivation in vivo. The bacteriophage P1 protein Cre (Cyclization Recombination) catalyzing recombination between two 34 bp loxP sites is the most-used recombinase system (Branda and Dymecki, 2004; Metzger and Feil, 1999). Two transgenic mouse lines are required for the Cre-loxP system, one mouse line expressing Cre recombinase under the control of a specific gene regulatory promoter and another mouse line carrying a floxed (loxP flanked) allele of the interest
gene (Kwan, 2002). In offspring derived from an inter-cross of these two mouse lines, the gene of interest is deleted only in specific cells where the Cre is expressed. Cholesterol side-chain cleavage enzyme (cytochrome P450scc) plays a key role in the synthesis of steroid hor-mones, which is encoded by CYP11A1 gene (Guo et al., 2003; Miller, 1988). P450scc converts the precursor cho-lesterol into pregnenolone which is then changed into different steroids by the sequential action of numerous enzymes. P450scc is present primarily in adrenal glands and gonads for the major production of steroid hor-mones. In addition, it is also found in the placenta (Durkee et al., 1992), the primitive gut (Keeney et al., 1995), and the brain (Mellon and Deschepper, 1993). The levels of CYP11A1 expression in the brain are much lower than that in adrenal glands (Furukawa et al., 1998; Sanne and Krueger, 1995; Watzka et al., 1999). The expression of CYP11A1 in adrenals and gonads is stimu-lated by specific tropic hormones secreted from the pitu-itary gland via cAMP-dependent pathway (Guo et al., 2003). Orphan nuclear receptor steroidogenic factor-1 (SF-1; NR5A1) is a crucial transcriptional regulator of steroidogenic genes in adrenals and gonads (Parker and Schimmer, 1997). Little information is available on the transcriptional regulation of CYP11A1 in the brain.
The 2.3-kb 50-flanking sequence of the human CYP11A1 contains all known regulatory elements, in-cluding two functional SF-1 binding sites around 40 and 1600, an upstream cAMP-responsive sequence at 1540/1640 and an enhancer sequence AdE at
* Correspondence to: Meng-Chun Hu, Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei 100, Taiwan, Repub-lic of China.
E-mail: [email protected]
Contract grant sponsor: National Science Council, Contract grant num-ber: NSC93-2320-B-002-064.
yHsu-Shui Wu and Hui-Ting Lin contributed equally to this work.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/dvg.20266
1932/1822 (Chou et al., 1996; Guo et al., 1994; Hum et al., 1993; Inoue et al., 1991). This 2.3-kb fragment has been shown to drive simian virus 40 (SV40) T antigen expression and lead to adrenal tumor development in vivo (Mellon et al., 1994). Furthermore, the 2.3- or 4.4-kb fragment was used to control the expression of LacZ gene in transgenic mice. The LacZ was expressed specifically in adrenals, testes, and ovaries (Hu et al., 1999). These results indicate that the 2.3-kb promoter possess the ability to drive the transgene expression spe-cifically in adrenal glands and gonads. However, the expression of transgene in the brain and placenta has not been demonstrated.
The present study is to generate SCC-Cre transgenic mice in which the Cre recombinase expression is under the control of the human CYP11A1 promoter. To increase the level of Cre expression, we selected the codon-improved Cre (iCre) gene (Shimshek et al., 2002), in which the modified codon usage was optimal for eukaryotes, instead of using the bacteriophage P1-derived Cre recombinase. In addition, the insulation sequence derived from chicken b-globin LCR (Chung et al., 1993) was introduced into transgene constructs to prevent the position effect (Recillas-Targa et al., 2004). In this report, we show that this human CYP11A1 pro-moter targets Cre recombinase expression to adrenals, testes, and ovaries. Furthermore, the expression of Cre is found in the brain. The results were the first to eluci-date that the 4.4-kb promoter is able to direct reporter expression in the brain.
RESULTS
Generation of SCC-Cre Transgenic Mice
The 4.4-kb of 50-flanking region of the human CYP11A1 gene was fused to the iCre gene and SV40 polyA sequence to generate the construct SCC-iCre. To obtain an appropriate level of transgene expression, two copies of the HS4 insulator sequences derived from chicken b-globin LCR were inserted into the CYP11A1 promoter/iCre/SV40 polyA cassette at the upstream or downstream in reverse direction as shown in Figure 1. After pronuclear injection of constructs, founder mice were identified by polymerase chain reaction (PCR) anal-ysis. Four independent SCC-Cre transgenic lines were generated from two constructs, respectively.
To test the efficiency of Cre recombinase activity in transgenic mice, SCC-Cre mice were crossed to the ROSA26R reporter line to create double transgenic SCC-Cre/R26R offspring. ROSA26R mice contain the LacZ gene that is only expressed by Cre-mediated recombina-tion (Soriano, 1999). Thus, the expression of Cre can be elucidated by X-gal staining for b-galactosidase activity. Steroidogenic tissues including adrenal glands, testes, and ovaries from SCC-Cre/R26R mice were assayed by b-galactosidase staining to determine the Cre activity. Three of four established mouse lines for each transgene construct displayed Cre activity in these tissues. Table 1 summarizes the expression level of the transgene in the examined organs. In addition to expression in adrenals and gonads, four transgenic mouse lines also exhibited Cre expression in the brain, in which the expression of the endogenous CYP11A1 is much lower than that in the adrenal. Our results indicate that these two trans-gene constructs, with opposite orientations and loca-tions of insulator fragments relative to the CYP11A1 pro-moter, have no difference in the ratio of transgene expression. The mouse line Nos.16 and 30 exhibited higher expression levels and consistent expression pat-tern in most of tissues analyzed.
Expression of Cre in Adrenal Glands and Gonads To determine the transgene expression pattern, tissue sections of SCC-Cre/R26R mice were examined by b-ga-lactosidase staining. The b-galactosidase activity was
FIG. 1. Schematic representation of SCC-Cre transgene con-structs. The plasmid contains a 4.4-kb fragment of 50-flanking region from human CYP11A1 gene placed in front of the iCre gene and the SV40 polyadenylation sequence (polyA). Two copies of 1.2-kb fragments containing the HS4 element from chicken b-globin gene were cloned in the upstream or downstream of the CYP11A1 promoter/iCre/SV40 polyA cassette. The pInSCC4.4-iCre and pSCC4.4-iCreIn plasmids contain the HS4 elements in the forward and reverse orientations, indicated by the arrow, respectively.
Table 1
Cre-Recombined LacZ Expression in SCC-Cre Mouse Lines Line
Transgene construct
Adrenal
cortex Testis Ovary Brain 16 InSCC-iCre þþþ þþþþ þþþþ þþþþ 20 InSCC-iCre þþ þþ þþ – 30 InSCC-iCre þþþþ þþþþ þþþþ þþþþ 11 SCC-iCreIn þ þþþ þþ – 14 SCC-iCreIn þþ þþ þþ þþþ 41 SCC-iCreIn þþþþ þþþþ þþþ þþþ Expression levels of LacZ gene were determined by b-galactosi-dase staining in various tissues from SCC-Cre/R26R mice at the ages of 2–6 months.
observed in the adrenal cortex and was not detected in the medulla of SCC-Cre/R26R mice. Transgenic line Nos.16, 30, and 41 showed the LacZ staining in most cells of all adrenocortical zones (Fig. 2a). These results were consistent with the endogenous Cyp11a1 expres-sion in the adrenal cortex (Fig. 2g). A special expresexpres-sion pattern was found in line No. 11. The b-galactosidase staining was limited to the innermost X zone of the adre-nal cortex, with only a few staining in the zona fascicu-lata and glomerulosa (Fig. 2b). Because the X zone degenerates after puberty in male mice (Deacon et al., 1986), rare blue staining was seen in the adrenal cortex of the adult male line No. 11 (Fig. 2c). Similar result also was found in a mouse line of previous SCC-LacZ trans-genic mice (Hu et al., 1999).
In the testis, all mouse lines exhibited the b-galactosi-dase staining in the Leydig cells that lie in the space between tubules (Fig. 2d). A similar expression pattern was detected for endogenous Cyp11a1 mRNA by in situ hybridization (Fig. 2h). In the ovary, the extent of b-galac-tosidase expression varied among different mouse lines. Most lines showed high levels of b-galactosidase activity in the corpora lutea and stromata. Moreover, the promi-nentb-galactosidase staining was detected in theca cells of line No. 30 (Fig. 2e) and in granulosa cells of line No. 16 (Fig. 2f). These correlate with in situ localization of Cyp11a1 mRNA in corpora lutea, stromata, theca cells, and also in granulosa cells of some follicles (Fig. 2i).
To determine spatial and temporal patterns of Cre expression during the development, we examined the b-galactosidase activity at different stages of embryos by whole mount staining. Embryos of SCC-Cre/R26R from transgenic line Nos. 16, 30, and 41 gave similar
expres-sion patterns, and embryos of SCC-Cre and ROSA26R lit-termates did not show blue staining in any tissues. A slight b-galactosidase staining was first detected in the adrenal primordia on embryonic day 11.5 (data not shown). By E12.5, the signal was intensified and the prominent b-galactosidase staining was constantly occurred in adrenal glands in the following developmen-tal stages (Fig. 3). In gonads, weak X-gal staining was ini-tially observed on E12.5 (Fig. 3a,d) and revealed no obvious sex differences. The b-galactosidase activity remained low in embryonic ovaries on E15.5 (Fig. 3b). On the contrary,b-galactosidase staining became signifi-cant through the entire testis beginning at E14.5 embryos (Fig. 3e). By E15.5, a high level of b-galactosi-dase activity was visible in descended testes (Fig. 3f). A similar expression pattern of the endogenous Cyp11a1 on E14.5 embryo was seen by whole-mount in situ hybridization analysis. High levels of Cyp11a1 mRNAs were present in the developing adrenal and testis (Fig. 3c,g); however, the expression of Cyp11a1 mRNAs was undetected in the ovary (Fig. 3c).
In addition to the classical sites for steroids synthesis, theb-galactosidase activity was also found in some non-steroidogenic tissues. Tissue sections showed that dis-tinct staining was observed in the developing gut and heart (Fig. 3h,i). Although placenta is a steroidogenic tis-sue, the expression of Cre in the placenta could not be detected by whole-mount or tissue-section staining with X-gal.
Expression of Cre in the Brain
Because the gene expression of CYP11A1 is very low in the brain, the analysis of CYP11A1 promoter function
FIG. 2. Cre-mediated b-galacto-sidase expression in adrenal glands and gonads of SCC-Cre/ R26R mice. Tissue sections from transgenic line No. 30 (a, d, and e), No. 11 (b and c) and No. 16 (f) at 6–12 weeks were stained with X-gal. Sections (g–i) were hybri-dized with Cyp11a1 riboprobes to show the endogenous gene exp-ression. Tissues, including ad-renal glands (a–c and g), testes (d and h), and ovaries (e, f, and i), were collected at 6–12 weeks of age. The arrowhead indicates theca cells and the arrow indi-cates granulosa cells. Scale bars ¼ 100 lm. C, adrenal cortex; CL, corpus luteum; f, follicle; L, Ley-dig cell; M, medulla; S, stroma; ST, seminiferous tubule. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.]
in the brain has not been demonstrated in vivo. To evalu-ate whether the 4.4-kb promoter contains regulatory sequence to drive the Cre gene expression in the brain, the whole brain tissue was sagittally divided into two parts from the middle and then stained with X-gal. The significant b-galactosidase staining was observed in the diencephalon (including epithalamic, anterior thalamic, and hypothalamic regions) and the tectum of the mid-brain, while the cerebral cortex and the cerebellum were largely unstained (Fig. 4a). Since the distribution of Cyp11a1 in the hippocampus has been extensively
ana-lyzed by in situ hybridization and immunohistochemical staining previously (Furukawa et al., 1998; Kimoto et al., 2001), we thus examined Cre activity in the hippocam-pus by tissue-section staining. Theb-galactosidase stain-ing was detected along the pyramidal cell layer in CA1– CA3 regions and the granule cell layer in the dentate gyrus (Fig. 4b). Similar pattern of the endogenous Cyp11a1 expression was reported in the hippocampus of rat brain (Furukawa et al., 1998; Kimoto et al., 2001). Theb-galactosidase activity was also found in the hypo-thalamus which is a distinct structure of diencephalon.
FIG. 3. Cre mediated b-galacto-sidase expression in embryonic tissues of SCC-Cre/R26R mice. Embryos from SCC-Cre/R26R mice at different gestational stages were stained for b-galactosidase activity (a,b, d–f, h,i) or assayed for the endogenous Cyp11a1 expres-sion by in situ hybridization (c and g). Wholemount adrenals and gonads stained with X-gal are shown in a, d, e (line No. 30), b (line No. 41), and f (line No. 16). Sagittal sections (h and i) of E15.5 embryo (line No. 30) were stained with X-gal and counterstained with Nu-clear Fast Red. Arrows in h and i indicate X-gal staining in the gut and heart, respectively. Ad, adrenal gland; H, heart; K, kidney; Lg, lung; Lr, liver; O, ovary; T, testis.
FIG. 4. Cre mediatedb-galactosidase expression in brains of SCC-Cre/R26R mice. The following brain parts were stained with X-gal, whole mount of adult brain that is cut mid-sagittally by hand (a), coronal sections through hippocampus (b) or hypothalamus (c), and whole mount embryonic brains (d–f). Arc, arcuate nucleus; CA, cornu ammonis; DG, dentate gyrus; DC, diencephalon; MB, midbrain; Py, pyrami-dal cell layer; TC, telencephalon; VMHDM, ventromedial hypothalamic nucleus dorsomedial part.
Theb-galactosidase activity was specifically identified in the dorsomedial part of ventromedial hypothalamic nu-cleus and arcuate nunu-cleus (Fig. 4c). The expression of endogenous Cyp11a1 in this region has not been reported previously.
During embryogenesis, b-galactosidase expression in the brain was detected as early as E10.5. The b-galactosi-dase activity was clearly observed in the diencephalon and the midbrain and was not seen in the telencephalon (Fig. 4d). As development proceeded, the telencephalon expanded bilaterally and continued to extend caudally so as to cover the diencephalon. As shown in Figure 4f, the intense staining within the diencephalon was located beneath the telencephalon on E14.5. In addition, the embryos displayed a dispersed b-galactosidase ex-pression in the midbrain at earlier stages (Fig. 4d,e). As the midbrain continued to differentiate, the b-galactosi-dase staining appeared to be condensed with distinct structure on E14.5 (Fig. 4f). Both spatial and temporal distributions of b-galactosidase expression in the devel-oping embryos were similar among three examined transgenic mouse line Nos. 16, 30, and 41.
DISCUSSION
The aim of this study was to generate transgenic mice expressing Cre recombinase under the control of the human CYP11A1 promoter. We showed that the 4.4-kb 50-flanking sequence is able to drive Cre transgene expression specifically in adrenal glands, gonads, and the brain. To prevent the negative position effect and improve the transgene expression, an insulator element was added into our constructs. Two copies of insulator fragments from chicken b-globin HS4 element (Chung et al., 1993) were cloned at the 50or 30end of the trans-gene construct to test whether the location may influ-ence the effect of insulator. For each construct, four transgenic lines were established and three of them expressed the transgene, respectively. No significant dif-ference was noticed in the number of expressing lines and in the expression level for these two constructs. This demonstrates that the insulator improves the speci-ficity of transgene expression.
The 4.4-kb promoter has previously been linked to LacZ gene for transgenic mice generation (Hu et al., 1999). Consistent with the SCC-LacZ mice study, the Cre-mediated b-galactosidase expression was found in adrenals, testes, and ovaries but not in the placenta. The transgenic line Nos. 30 and 16 displayed the best Cre ac-tivity in the adrenal cortex, Leydig cells of testis, corpor lutea, stromata, and follicular cells of ovary. The expres-sion pattern of Cre correlates with the endogenous gene expression in these tissues. During embryogenesis, b-ga-lactosidase expression could be clearly detected in the developing adrenal at E12.5 and also in gonads at low levels. By E14.5, the developing testis abundantly expressed b-galactosidase. In addition, Cre expression was found in the nonclassic steroidogenic tissues, gut,
and heart. Previous studies have verified the expression of Cyp11a1 gene in the primitive gut during murine em-bryonic development (Keeney et al., 1995), but the physiological function is not clear. Our data elucidated that the 4.4-kb promoter was sufficient to drive gene expression in the embryonic gut.
Although the expression of CYP11A1 in the heart has not been reported, mRNA of downstream steroidogenic genes, Cyp11b1 and Cyp11b2, has been detected in the rat heart in which they can function for the production of cardiac aldosterone and corticosterone (Silvestre et al., 1998). We thus suspect that the endogenous CYP11A1 may exist in the heart for local steroids synthe-sis from the precursor cholesterol.
Some studies have reported that CYP11A1 mRNAs is widely expressed in the brain, but its amount is signifi-cantly lower than that in adrenals (Furukawa et al., 1998; Sanne and Krueger, 1995; Watzka et al., 1999). The inability to detect transgene expression in the brain of SCC-LacZ mice (Hu et al., 1999) may reflect the con-siderably low amount of endogenous CYP11A1 mRNAs. However, we detect Cre recombinase activity in the SCC-Cre transgenic brain. The Cre expression in the brain should be real, because three SCC-Cre transgenic lines (Nos. 16, 30, and 41) consistently displayed region-specific expression patterns of Cre gene in the adult and embryonic brains. Furthermore, this promoter specifi-cally drives Cre expression in CA1–CA3 regions and the dentate gyrus (Fig. 4b) of the hippocampus. This pattern of Cre expression correlated with the endogenous Cyp11a1 expression in the hippocampus of rat brain (Furukawa et al., 1998; Kimoto et al., 2001). These results demonstrated that the 4.4-kb promoter of the human CYP11A1 can indeed direct Cre transgene expression in the mouse brain. Since the distribution of CYP11A1 mRNA is not clearly determined in most of brain regions, our SCC-Cre transgenic mice can serve as useful tools in elucidating the profile of CYP11A1 expression in brain tissues.
Although CYP11A1 is widely present in the brain tis-sue, little is known regarding the transcriptional regula-tion of CYP11A1 gene in the brain. A report showed that the 2.5-kb of 50-flanking region from rat Cyp11a1 is transcriptionally active in rat C6 glioma cells, but not in other neural cells such as rat GC somatotrope and mouse GT1–7 neurosecretory cells (Zhang et al., 1995). Our data are the first in vivo experiment to demonstrate that the 4.4 kb regulatory region from human CYP11A1 is transcriptionally active in the mouse brain. In addition, this 4.4-kb fragment is able to direct transgene expres-sion in the embryonic brain. It indicates that the 4.4-kb 50-flanking sequence contains essential regulatory ele-ments for CYP11A1 expression in the brain. However, the Cre expression was undetected in some parts of the brain such as the cortex and cerebellum that express CYP11A1 (Sanne and Krueger, 1995; Ukena et al., 1998). These results suggest that CYP11A1 expression could be differentially regulated in various regions of the brain.
SF-1 is an important regulator modulating CYP11A1 expression in adrenals and gonads. In the brain, SF-1 has been identified in the ventromedial hypothalamic nu-cleus, predominantly present within the dorsomdial part, and in pituitary gonadotropes (Shinoda et al., 1995). Since SF-1 is not expressed in most brain regions that express CYP11A1 (Morohashi, 1999) and also not in rat C6 cells (Zhang et al., 1995), SF-1 is assumed not to be involved in the regulation of CYP11A1 transcrip-tion in the brain. However, in SCC-Cre transgnic mice, we found the Cre gene is expressed in the dorsomedial part of ventromedial hypothalamic nucleus (VMHDM, Fig. 4c), in which SF-1 expression has been described. It suggests that CYP11A1 may be colocalized with SF-1 in the VMHDM and, hence, SF-1 may play a role in activat-ing CYP11A1 expression in this region.
In summary, we have obtained SCC-Cre transgenic mice harboring Cre recombinase activity specifically in the tissues for steroids production, including adrenals, testes, ovaries, and the brain. Two transgenic lines, Nos. 16 and 30, are valuable for the investigation of gene function in these tissues using the Cre-loxP system. Our studies further demonstrate that the 4.4-kb 50-flanking sequence can sufficiently activate reporter gene expres-sion in the brain. The Cre/R26R assay system provides a useful approach for understanding the transcriptional regulation of the CYP11A1 gene in the brain.
MATERIALS AND METHODS Transgene Construction
A 1.09-kb HindIII/KpnI fragment of iCre gene excised from the plasmid pBlue.iCre (Shimshek et al., 2002) (generously provided by Dr. Rolf Sprengel) was cloned into the pUC19 vector containing SV40 polyadenylation sequence to generate the plasmid piCre-polyA. The 1.94 fragment containing iCre and SV40 polyA isolated from the piCre-polyA was fused to the human CYP11A1 pro-moter corresponding to the 50-flanking sequence 4400/þ55 (Hu et al., 1999) and termed phSCC4.4-iCre. Two copies of 1.2-kb fragment containing the HS4 element from chicken b-globin gene (Chung et al., 1993) were introduced into the plasmid phSCC4.4-iCre. The HS4-containing fragments were placed in front of the CYP11A1 promoter or at the 30 end of SV40 polyA signal in reverse direction to create plasmids pInSCC4.4-iCre and pSCC4.4-pInSCC4.4-iCreIn, respectively (Fig. 1).
Generation of SCC-Cre and SCC-Cre/R26R Mice Two 8.8-kb DNA fragments containing the 4.4-kb CYP11A1 promoter, iCre, SV40 polyA, and two-copies of HS4 elements were used for the production of SCC-Cre transgenic mice. One was excised from plasmid pInSCC4.4-iCre with SalI and EcoRI and the other was released from plasmid pSCC4.4-iCreIn with SalI diges-tion. The transgene fragments were purified and micro-injected into pronuclei of fertilized oocytes from C57BL/ 6J. These eggs were then transferred to pseudopregnant
females. Transgenic founders termed SCC-Cre were gen-otyped by PCR analysis and mated to C57BL/6J mice to establish transgenic lines.
The SCC-Cre transgenic mice were mated with ROSA26 mice (purchased from The Jackson Laboratory, Bar Harbor, Maine) to produce SCC-Cre/R26R double transgenic mice that were identified for the presence of iCre and LacZ gene by PCR reaction.
Genotyping
Genomic DNA was prepared from mouse tails or embryos and analyzed for the presence of the transgene by PCR using the following conditions: 30 cycles of 948C for 30 s, 568C for 30 s, 728C for 1 min, and a final extension at 728C for 7 min. A 315-bp fragment was amplified for iCre gene with primers: forward 50 -TCAA-CATGCTGCACAGGAG-30; reverse 50 -TCCTGCCAATGTG-GATCAG-30. A 391-bp fragment was amplified for LacZ gene with primers: forward 50 -TCGTCAGTATCCCCGTT-TACAG-30; reverse 50-CGGTAGTTCAGGCAGTTCAATC-30). Primers specific for Sry (forward 50 -AAGCGCCCCAT-GAATGCATT-30; reverse 50 -CGATGAGGCTGATATTTATA-30(Luo et al., 1994) were used to determine the sexes of the embryo by PCR.
Analysis ofb-Galactosidase Expression
Tissues or embryos were collected from SCC-Cre/ R26R mice and stained with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) to identify the expression of b-galactosidase. For whole-mount staining, tissues or embryos were fixed with 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 1 h at 48C, washed with PBS and incubated overnight in X-gal solution (1 mg/ml X-gal, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 1.5 mM MgCl2, 0.1% NP40, and 0.15 mg/ml chloroquine in PBS). For sectioning, adrenals, testes, and ovaries were placed in OCT compound and frozen at 208C. Embryos were fixed with 0.2% paraformaldehyde in PBS overnight at 48C, washed with PBS, cryoprotected with 15 and 30% sucrose in PBS, and then embedded in OCT at 208C. For sectioning of adult brains, mice were anesthetized with pentobarbital and fixed by perfusion with 2.5% paraformaldehyde in PBS. Following perfusion, brain tis-sues were removed and embedded in OCT at 208C. The frozen tissue blocks were cut with a cryostat, trans-ferred to glass slides and post fixed with 2% paraformal-dehyde and 0.2% glutaralparaformal-dehyde in PBS for 10 min. Slides were washed with PBS and stained with X-gal (1 mg/ml X-gal, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, and 1.5 mM MgCl2in PBS) in a moist chamber overnight. Slides were then rinsed with PBS and counterstained with Nuclear Fast Red (Sigma) or Neutral-red (Sigma) for 5 min. After washing with PBS, tissue slices were mounted with IMMUNO-MOUNT (Shandon).
In Situ Hybridization
Embryonic tissues were fixed with 4% paraformaldehyde in PBS for 16 h at 48C, washed with PBS, rinsed in 100%
methanol for several times, and stored in 100% methanol at 208C. Tissues were rehydrated through 75, 50, and 25% methanol/PBS for 20 min each. After PBST (0.1% Tween-20 in PBS) washes, tissues were treated with 5 lg/ml proteinase K for 30 min at 378C. Tissues were postfixed with 4% paraformadehyde/0.1% glutaralde-hyde in PBST for 20 min and then performed for prehy-bridization. For sectioning, tissues were prepared in two ways. Adrenals were directly embedded in OCT. Testes and ovaries were first fixed with 4% paraformaldehyde in PBS overnight at 48C, cryoprotected with 15 and 30% sucrose in PBS, and then embedded in OCT. Frozen sec-tions were fixed with 4% paraformaldehyde/PBS for 2 h. After washing in PBST, slides were treated with 10lg/ml proteinase K for 10 min at 378C and then postfixed with 4% paraformadehyde/PBST. Following postfixation, tis-sues or slides were hybridized with DIG-labeled ribop-robes as described previously (Hu et al., 1999). After hybridization, samples were incubated with 5% blocking reagent (Roche) for 2 h at room temperature and then reacted with antiDIG-AP (1:5000 dilution in 5% blocking reagent, Roche) at 48C overnight. After three washes in maleic acid buffer (0.15 M NaCl and 0.1 M maleic acid, pH 7.5) for 30 min each, samples were equilibrated with detection solution (0.1 M NaCl, 50 mM MgCl2, and 0.1 M Tris; pH 9.5) for 5 min three times and then stained with BM purple/TNBT solution (CHMICON) for 4–7 h until color appearance. Staining reaction was stopped by washing with PBST and then sections were mounted and tissues were stored in 4% paraformaldehyde at 48C. ACKNOWLEDGMENTS
The authors thank the Transgenic Core Facility at Aca-demia Sinica for the generation of the transgenic mice and Yi-Lin Chien for technical assistance.
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