Thick ascending limb-specific expression of Cre recombinase



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发表于 2009-4-21 13:39 |显示全部帖子

作者：Peter K. Stricklett, Deborah Taylor, Raoul D. Nelson, and Donald E. Kohan,作者单位：Divisions of 1 Adult and 2 Pediatric Nephrology, University of Utah School of Medicine, and 3 Salt Lake Veterans Affairs Medical Center, Salt Lake City, Utah 84132

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      【摘要】
   Evaluation of thick ascending limb (TAL) function has been hindered by the limited ability to selectively examine the function of this nephron segment in vivo. To address this, a Cre/loxP strategy was employed whereby the Tamm-Horsfall (THP) promoter was used to drive Cre recombinase expression in transgenic mice. The THP gene was cloned from a mouse genomic library, and 3.7 kb of the mouse THP 5'-flanking region containing the first noncoding exon of the THP gene were inserted upstream of an epitope-tagged Cre recombinase (THP-CreTag). THP-CreTag transgenic mice were bred with ROSA26-enhanced yellow fluorescent protein (eYFP) mice (contain a loxP-flanked "STOP" sequence 5' to eYFP), and doubly heterozygous offspring were analyzed. THP and eYFP were expressed in an identical pattern with predominant localization to the renal outer medulla without expression in nonrenal tissues. eYFP did not colocalize with thiazide-sensitive cotransporter (distal tubule) or neuronal nitric oxide synthase (macula densa) expression. THP mRNA expression was detected only in kidney, whereas CreTag mRNA was also present in testes. These data indicate that THP-CreTag transgenic mice can be used for TAL-specific gene recombination in the kidney.
      【关键词】 TammHorsfall uromodulin Crelox gene knockout
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CONVENTIONAL GENE KNOCKOUT strategies have resulted in a wealth of developmental, physiological, and pathophysiological information. Despite this, whole animal gene disruption often leads to interpretative difficulties. For example, the targeted gene may be essential to normal development, precluding analysis of its functional role in adulthood. In addition, determining the biological role of a targeted gene in a specific organ may be confounded by functional changes in other organs or organ systems. At an even finer level, evaluating the function of a gene in a particular cell type using conventional gene-targeting methods is problematic. These issues are particularly germane to gene-targeting strategies used to examine renal function. The kidney contains at least 27 different cell types, many of whose function impacts that of other renal cells. It is apparent, therefore, that more specific means of targeting gene disruption are necessary to selectively evaluate the role of that particular gene product in a given renal cell type in vivo.
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Cell-specific gene targeting has been attained primarily by exploiting the Cre/loxP recombinase system. The Cre/loxP system was originally described in bacteriophage P1 where it was observed to cause site-specific DNA recombination ( 12 ). The two essential components are 1 ) loxP, a 34-bp DNA sequence containing two 13-bp inverted repeats and an asymmetric 8-bp spacer region and 2 ) Cre recombinase, a 343-amino acid monomeric protein that mediates recombination. In the presence of Cre recombinase, two loxP sites (in the same orientation) recombine resulting in excision of the intervening piece of DNA. This system can be used for cell-specific gene targeting in vivo. One mouse line (floxed line) is developed, using homologous recombination in embryonic stem cells, in which a region within the targeted gene that is essential to its function has been flanked by loxP sites. A second transgenic mouse line, generated by standard oocyte injection techniques, contains a cell-specific promoter-driving expression of a Cre recombinase transgene. Mating of these two lines yields offspring with Cre recombinase-mediated gene disruption occurring only in those cells in which the promoter is active.

Two renal cell-specific Cre recombinase expressing mouse lines have been reported: a collecting duct principal cell-specific line using the aquaporin-2 promoter ( 8 ) and a podoctye-specific line using the nephrin promoter ( 4 ). In this study, we describe the development of a third renal cell-specific Cre recombinase-expressing line. This line uses the Tamm-Horsfall (THP) promoter to achieve thick ascending limb (TAL)-specific Cre recombinase expression. At the outset of these studies, the THP promoter was chosen because THP had been localized by immunostaining exclusively to the TAL and early distal convoluted tubule ( 10 ) and because THP is produced in abundance (reviewed in Ref. 7 ). Subsequently, the report that the mouse THP promoter (3.0 kb of the proximal 5'-flanking region) was sufficient to drive expression of a green fluorescent protein specifically in the TAL and early distal convoluted tubule in transgenic mice ( 14 ) lent credence to this approach. We describe herein that the THP promoter driving Cre recombinase causes TAL-specific targeted gene recombination in mice.* \( A8 W: W* U: g- o

METHODS# A% A4 @0 B$ q6 [8 x
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Isolation of genomic clone containing the mouse THP promoter. A mouse genomic library was generated using bacterial artificial chromosomes (BACs) that contain inserts of mouse ES cell 129/SVJ genomic DNA of 65-100 kb (Genome Systems, St. Louis, MO). The library was screened using a probe that was generated with oligonucleotide primers recognizing the 3'-end of the mouse THP promoter (forward: 5'-GTC CAG TTC AGG AGT GTC CAG A-3'; reverse: 5'-GGA CTG GTC CAT TCA CAA GTA AG-3'). These primers were designed from a sequence obtained from the plasmid pBT-NK (gift from Satish Kumar, University of Oklahoma), which contains the 3'-end of the mouse THP promoter. Positive BAC clone DNA was subjected to enzymatic digestion for Southern blot analysis using the same PCR probe. A genomic fragment of 12.5 kb from Avr II digestion was cloned into the Spe I site of pBluescript KSII (Stratagene, La Jolla, CA), which was mutated at the Sma I site (pBS-THP).
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Construction of THP-CreTag transgene. The transgene mTHP-CreTag was constructed by first modifying pBS-THP at the 3'-end of the THP gene fragment. This region contains the first noncoding exon as well as a portion of the first intron of the THP gene and was modified to create a fusion of the transcriptional start/exon1 of the THP gene with the Cre recombinase expression vector pBS-CreTag ( 8 ). PBS-CreTag contains the Cre recombinase gene that is modified at the NH 2 -terminal end to include an 11-amino acid herpes simplex virus (HSV) glycoprotein D epitope tag (QPELAPEDPED). pBS-THP was digested with Sma I/ Eco RI, and the resulting 2.5-kb fragment was cloned into pBluescript KSII. The Eco RI site is 100 bp 5' to the translational start site of THP gene. This plasmid (pBS-THP2.5) was then digested and a modified sequence was inserted 3' to the Eco RI site. The modified sequence was made using complementary oligonucleotides and Klenow DNA polymerase to make double-strand DNA. The manufactured sequence is identical to endogenous with the exception of including Not I and Xho I sites within exon I. The oligonucleotide was digested with Eco RI/ Xho I and cloned into pBS-THPp2.5. pBS-THP2.5 was then digested with Sma I/ Xho I, and a modified fragment was reinserted into a Sma I/ Xho I-digested pBS-THP. At this point, we were never able to isolate full-length clones that had not undergone recombination within the promoter region. Consequently, a clone containing 3.7 kb of the THP promoter was identified, and its sequence was confirmed (University of Utah Core DNA Sequencing Facility). The 3.7-kb THP gene fragment was cloned into pBluescript KSII, digested with Not I, blunted with Klenow, and cloned into pBS-CreTag that had been digested with Sma I. The final transgene THP-CreTag was sequenced in its entirety before injection.. g2 B5 {. H! L1 k! f" |" l! V
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Generation and breeding of THP-CreTag transgenic mice. Transgenic mice containing THP-CreTag were created by Xenogen Biosciences (Cranbury, NJ). The transgene was removed from the vector backbone with Not I/ Xho I digestion followed by gel electrophoresis. The transgene was excised from gel, digested with -Agarase (New England Biolabs, Beverly, MA), purified over an Elutip minicolumn (Schleicher & Schuell, Keene, NH), and resuspended in sterile injection buffer (10 mM Tris-base, pH 7.5, 0.25 µM EDTA). The linearized transgene was injected into the pronuclei of C57BL/CBA single-cell embryos, and the injected embryos were transferred into pseudopregnant mice according to standard procedures at Xenogen. Pups were shipped to Utah and analyzed for the presence of the transgene by PCR amplification of tail DNA. Individual founders were each bred to nontransgenic C57BL/CBA mice. F 1 and F 2 animals from each founder line were identified by PCR of tail DNA and used for analysis.
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Identification of THP-CreTag transgenic animals. Tail DNA was isolated according to standard methods, and the THP-CreTag transgene was detected by PCR amplification using oligonucleotides primers (forward: 5'- GTC CAG TTC AGG AGT GTC CAG A-3' located 552 bp 5' to the translational start site of the THP gene and reverse: 5'-GCG AAC ATC TTC AGG TTC TGC GG-3' located within the CreTag gene) that span 864 bp of the junction between the THP promoter and CreTag reporter. In some samples, normal mouse DNA with 1-100 copies/cell equivalent of the transgene DNA was run as a control to estimate copy number. Equal loading and amplification efficiency of genomic DNA were controlled for by amplification for GAPDH (5'-CCT TCA TTG ACC TCA ACT ACA TGG-3' AND 5'-GCA GTG ATG GCA TGG ACT GTG GT-3'). Products were electrophoresed through 2% agarose and visualized by ethidium bromide staining and ultraviolet illumination.

RT-PCR determination of THP, CreTag, and GAPDH mRNA expression. RNA was prepared from transgenic mouse organs with the acid phenol method ( 3 ). Total RNA (2.5 µg) from organs was reverse transcribed using oligo(dT) 12-18 and Superscript II according to manufacturer procedure (GIBCO/BRL, Gaithersburg, MD). The following oligonucleotide primers were used to PCR amplify THP, CreTag, and GAPDH: THPF 5'-AGG GCT TTA CAG GGG ATG GTT G-3' and THPR 5'-GAT TGC ACT CAG GGG GCT CTG T-3' amplified 441 bp of mouse THP cDNA; CreTagUP 5'-GAC TCT GGT CAG AGA TAC CTG G-3' and SV40LateR 5'-T 24 GTT GTT AA-3' amplified 411 bp of CreTag cDNA; and GAPDH primers (described above) amplified 442 bp of cDNA. All RT-PCR reactions were carried out with and without reverse transcriptase to establish whether cDNA or genomic DNA was being amplified. A negative control without template was also run with each set of reactions to demonstrate that there was no contamination of the PCR reaction. Products were electrophoresed through 2% agarose and visualized by ethidium bromide staining and UV illumination. All PCR products were directly sequenced using a dye terminator cycle-sequencing system with Amplitaq DNA polymerase, FS (Perkin Elmer, Norwalk, CT) in conjunction with an ABI fluorescent sequencing system (Core DNA Sequencing Facility).
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Generation and identification of THP-CreTag/ROSA26-enhanced yellow fluorescent protein mice. THP-CreTag mice were bred with ROSA26-enhanced yellow fluorescent protein (eYFP) reporter mice [provided by Frank Costantini, Columbia University, NY ( 11 )] to visualize the degree and specificity of Cre expression. Animals that were heterozygous for both THP-CreTag and the ROSA26-eYFP alleles were identified by tail DNA analysis using THP-CreTag primers (864-bp product as described above) and ROSA26-eYFP primers: forward: 5'-GGT TGA GGA CAA ACT CTT CGC-3' located within the ROSA26 locus and reverse: 5'-AAC TTG TGG CCG TTT ACG TCG-3' located within the eYFP gene (2.5-kb product).+ @4 T+ A: ^' |8 G$ y5 r

Immunofluorescent labeling of THP in the kidney. Mice heterozygous for THP-CreTag and ROSA26-eYFP underwent cardiac perfusion with PBS followed by perfusion with 2% paraformaldehyde (PFA) in PBS. Kidneys were excised and fixed for either 1 or 24 h (latter time period used only in some eYFP- and THP-staining studies) at 4°C in 2% PFA/PBS. Kidneys were then rinsed with PBS, imbedded in 3% agarose, and 200-µm sections were made using a vibratome (model OTS4000, Electron Microscopy Sciences, Fort Washington, PA). Sections for all staining procedures were washed in PBS, placed in 50 mM NH 4 Cl in PBS at room temperature for 30 min, rinsed in PBS, and washed several times with PBS-SG (0.2% bovine serum albumin, 0.5% saponin, and 0.2% gelatin in PBS). For THP staining, sections were placed in PBS-SG-D (PBS-SG containing 10% normal donkey serum) for 30 min, followed by incubation in a 1:100 dilution of sheep-anti-uromucoid (Biodesign International, Saco, ME) in PBS-SG-D at 4°C overnight. Sections were then rinsed in PBS-SG-D and incubated with a 1:100 dilution of CY5-conjugated donkey-anti-sheep antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS-SG-D at 4°C overnight. For neuronal nitric oxide synthase (nNOS) staining, sections were placed in PBS-SG-G (PBS-SG containing 10% normal goat serum) for 30 min, followed by incubation in a 1:25 dilution of rabbit-anti-nNOS (Zymed Laboratories, San Francisco, CA) in PBS-SG-G at 4°C overnight. Sections were then rinsed in PBS-SG-G and incubated with a 1:100 dilution of CY5-conjugated goat-anti-rabbit antibody (Zymed) in PBS-SG-G at 4°C overnight. For thiazide-sensitive cotransporter (TSC) staining, sections were placed in 5% skim milk-PBS for 30 min, followed by incubation in a 1:100 dilution of rabbit anti-mouse TSC (kindly provided by Dr. David Ellison, Oregon Health Sciences University, Portland, OR) ( 2 ) at 4°C overnight. Sections were then rinsed in 5% skim milk-PBS and incubated with a 1:100 dilution of CY5-conjugated goat anti-rabbit antibody in 5% skim milk-PBS at 4°C overnight. All sections were subsequently washed in PBS, mounted on slides using Vectashield (Vector Laboratories, Burlingame, CA), and viewed with either a Nikon fluorescent microscope (see Fig. 3 ) or a Zeiss confocal microscope (see Fig. 4 ) equipped with a CCD camera. Images were digitally acquired with Visis Smartcapture version 2.4.

Fig. 3. THP and eYFP expression in kidneys from mice heterozygous for both THP-CreTag and ROSA26-eYFP. Left : THP fluorescence (red); right : eYFP fluorescence (yellow/green). Images on the left and right are from adjacent 200-µm renal sections and are taken at x 40 ( top ) and x 400 ( bottom ). Note that THP and eYFP are expressed in a similar pattern and are localized primarily to the outer medulla. Photomicrographs are representative of those obtained from 5 offspring derived from line 1 THP-CreTag mice.2 j! d% h$ _0 I0 F  T

Fig. 4. Neuronal nitric oxide synthase (nNOS), thiazide-sensitive cotransporter (TSC), THP, and eYFP expression in kidney from mice heterozygous for both THP-CreTag and ROSA26-eYFP. Panels are arranged vertically with all panels in a column showing images obtained from the same section. Top : (red) staining for nNOS ( A ) and TSC ( D ) in cortex and THP ( G ) in outer medulla. Bottom ( C, F, I ): eYFP expression in the corresponding sections. Middle ( B : nNOS eYFP; E : TSC eYFP; H : THP eYFP): merged images from top and bottom. All images are from 200-µm renal sections and are taken at x 100 except for G, H, and I, which were taken at x 200. Note that nNOS and TSC do not colocalize with eYFP, whereas THP and eYFP are expressed by the same tubules. Photomicrographs are representative of those obtained from 5 offspring derived from line 1 THP-CreTag mice.& @3 {5 g% B) N* X

Fluorescent localization of eYFP expression in organs. Mice heterozygous for ROSA-eYFP alone or heterozygous for both THP-CreTag and ROSA26-eYFP underwent cardiac perfusion with PBS followed by perfusion with 2% PFA in PBS. Kidney, liver, skeletal muscle, heart, lung, brain, testes, vas deferens, ovary, spleen, stomach, intestine, and thymus were excised and fixed overnight at 4°C in 2% PFA/PBS. Organs were imbedded in 3% agarose, sectioned as described above, and eYFP was visualized using an eYFP filter with the system described above; positive cells fluoresced yellow/green.; E! a( M: s2 O6 H5 ]# O

RESULTS

Transgene design. To achieve TAL-specific gene expression in the kidney, a transgene with the THP 5'-flanking region driving expression of a modified Cre recombinase (CreTag) was designed ( Fig. 1 ). The CreTag cassette consisted of an SV40 nuclear localization signal on the NH 2 terminus of Cre recombinase, an 11-amino acid epitope tag, derived from HSV glycoprotein D on the COOH terminal to facilitate immunodetection of Cre recombinase, and an intronless polyadenylation signal from the SV40 late region for efficient expression ( 8 ). CreTag contains 362 amino acids with a predicted molecular mass of 41 kDa and has been shown to exhibit loxP site-specific recombinase activity in vitro and in vivo ( 8 ). A transgene was then constructed with 3.7 kb of the mouse THP 5'-flanking region containing the first noncoding exon of the THP gene ( 9 ). Sequencing of the THP construct confirmed it was identical to the published mouse THP promoter sequence ( 14 ). The final construct, containing the 3.7-kb THP fragment and CreTag, was termed THP-CreTag and is illustrated in Fig. 1.
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Fig. 1. Schematic of mouse Tamm-Horsfall protein (THP) promoter epitope-tagged Cre recombinase DNA construct. The THP fragment consists of 3.7 kb of the mouse THP 5'-flanking region and contains the first noncoding exon as well as a portion of the first intron of the THP gene. Cre recombinase has a nuclear localization sequence on the NH 2 terminus and a herpes simplex virus glycoprotein D epitope tag on the COOH terminus. An SV40 polyA signal is present for efficient expression.2 M3 h6 H) \9 P: z# F; Z/ M
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Generation of THP-CreTag transgenic mice. Eleven independent founders were obtained following pronuclear injections of the THP-CreTag transgene. PCR analysis of F 1 tail DNA demonstrated that only six of the founders transmitted the transgene. These six founders were used for subsequent analysis. The transgene copy number was not analyzed in each founder line at this point because it seemed most relevant to first determine which line conferred the most specific and greatest degree of recombination.

Cre recombinase activity in THP-CreTag/ROSA26-eYFP mice. THP-CreTag mice from each of the six founder lines were bred with ROSA26-eYFP reporter mice, and offspring heterozygous for both THP-CreTag and the ROSA26-eYFP alleles were identified by tail DNA analysis. These mice were then used for analysis of Cre recombinase-mediated recombination. ROSA26-eYFP reporter mice express eYFP in the presence of Cre recombinase as a result of site-specific recombination at loxP sites flanking a STOP sequence ( Fig. 2 ). Thus only cells in which Cre recombinase is active should exhibit yellow/green fluorescence.
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Fig. 2. Schematic of Cre recombinase (CreTag)-mediated recombination in the ROSA26-enhanced yellow fluorescent protein (eYFP) reporter genomic site. eYFP has been inserted immediately downstream of the ubiquitous promoter ROSA26. eYFP is not expressed because a loxP-flanked "STOP" sequence prevents read-through of DNA polymerase. In the presence of CreTag, the loxP sites recombine, eliminating the "STOP" sequence and permitting expression of eYFP.

Kidney sections from THP-CreTag/ROSA26-eYFP mice were examined for eYFP activity first. Mice derived from the six different THP-CreTag founder lines exhibited variable fluorescence intensity; however, all mice demonstrated activity primarily in the outer medulla. Much less eYFP activity was observed in the cortex and none was present in the inner medulla. The activity in the cortex consisted of several tubules extending out of the outer medulla; blood vessels and glomeruli were negative. One THP-CreTag founder line ( line 1 ) yielded substantially greater eYFP activity in the outer medulla after breeding with ROSA26-eYFP mice than other lines; this THP-CreTag line was used for subsequent analysis. Detailed examination of eYFP fluorescence in kidneys from at least five offspring derived from the line 1 founder revealed a yellow/green fluorescence pattern strongly suggestive of TAL labeling ( Fig. 3 ). As stated above, no eYFP was detected in the inner medulla and scattered tubules were positive in the cortex. Note that these photomicrographs are from sections fixed for 24 h in 2% PFA; this enhanced eYFP detection. Kidney sections were also stained for THP expression to confirm the cellular pattern of expression of eYFP. Low ( x 40)- and high ( x 100)-power photomicrographs ( Fig. 3 ) demonstrate a similar distribution (low power) and intracellular pattern (high power) of THP staining (red fluorescence) and eYFP activity (yellow/green). These THP photomicrographs were taken using a fluorescent microscope from sections that were fixed for only 1 h in 2% PFA (short incubation time optimizes THP detection). The disadvantage of viewing separate sections was clearly that precise colocalization of eYFP and THP (or other epitopes) fluorescence could not be attained. Consequently, studies were performed on eYFP colocalization with THP, nNOS, and TSC expression using the same sections wherein all tissue was incubated with 2% PFA for 1 h and viewed by confocal microscopy. The tradeoff, as can be seen in Fig. 4, is that the eYFP signal is not as bright or sharp as that described above. With the use of these latter procedures, eYFP and THP clearly localized to the same nephron segments ( Fig. 4, G - I ). In addition, nNOS ( Fig. 4, A - C ) and TSC ( Fig. 4, D - F ) did not colocalize with eYFP, indicating that Cre recombinase was not expressed in macula densa (nNOS) ( 1 ) or distal tubule (TSC) ( 2 ). Finally, it was not possible to quantitatively assess the percentage of THP-positive cells that expressed eYFP; however, examination of adjacent sections suggests that at least 70-80% of TAL cells expressed eYFP, if not more.
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eYFP was assessed in a panel of organs to evaluate the specificity of Cre recombinase activity under control of the THP promoter. Sections (200 µm) from liver, skeletal muscle, heart, lung, brain, testes, vas deferens, ovary, spleen, stomach, small intestine, large intestine, and thymus were obtained from at least three different THP-CreTag/ROSA26-eYFP, THP-CreTag alone, or ROSA26-eYFP mice (THP-CreTag containing mice all derived from the line 1 founder). There was no significant difference in eYFP expression between nonrenal organs from THP-CreTag/ROSA26-eYFP, THP-CreTag, and ROSA26-eYFP mice; liver and brain derived from all mouse lines had a minimal amount of background staining and other organs were negative (data not shown).# c8 f# Z* T2 c" [3 U1 c2 e; M5 C

RT-PCR determination of THP, CreTag, and GAPDH mRNA expression in THP-CreTag transgenic mice. To further confirm the tissue pattern of expression of CreTag, mRNA levels of THP, CreTag, and GAPDH were assessed in organs derived from THP-CreTag transgenic mice. Primers were used that spanned an intron in THP and GAPDH. The CreTag primers were designed to uniquely amplify CreTag mRNA even though the transgene is intronless. This was achieved, as previously described ( 8 ), by engineering the downstream primer to anneal to the polyA mRNA tail and a 6-bp anchor sequence immediately 5' to this site. THP mRNA was detected only in kidneys of line 1 THP-CreTag mice and not in brain, heart, thymus, lung, liver, spleen, stomach, small intestine, large intestine, ovary, testes, vas deferens, or skeletal muscle ( Fig. 5 ). CreTag mRNA was detected in the kidney and testes ( Fig. 5 ). A very faint band for CreTag mRNA was also seen in the brain.7 G3 v4 d0 N( J$ O

Fig. 5. RT-PCR determination of THP, CreTag, and GAPDH mRNA in organs obtained from mice heterozygous for THP-CreTag. The gel is representative of results obtained from 3 offspring derived from line 1 THP-CreTag mice. Sm., small; Lg., large./ O: _( k- Z2 ~5 K: v

Determination of THP-CreTag transgene copy number. Having found that line 1 THP-CreTag mice produce Cre recombinase selectively within the kidney in a pattern indicative of TAL-specific expression, the copy number of CreTag transgene was determined. Line 1 THP-CreTag mice contained 2 copies/cell equivalent compared with standards of transgene mixed with normal mouse genomic DNA, indicating chromosomal insertion of one copy of the transgene. As expected, the transgene was transmitted according to Mendelian genetics.

DISCUSSION. r4 R" ?6 u% B$ E& |7 Y

The present study describes the development of transgenic mice expressing Cre recombinase selectively within the TAL in the kidney. Although not possible to be precisely quantitative, labeling of adjacent tissue sections suggests that Cre recombinase is expressed in a high (at least 70-80%) percentage of TAL cells. Importantly, these studies demonstrated that mice transgenic for THP-CreTag not only express Cre recombinase in a TAL-specific manner but that genomic recombination does occur in TAL cells. Furthermore, these studies indicate that Cre recombinase is not expressed in macula densa or distal tubule cells. Although there has been some controversy over the presence of THP in macula densa and/or distal tubule, detailed studies by Hoyer and Seiler (reviewed in Ref. 5 ) strongly suggest that THP is only synthesized by the TAL and its presence in macula densa or distal tubule is entirely confined to the lumen. Hence, THP-CreTag mice permit TAL-specific expression of a biologically active Cre recombinase.1 F. U9 ?6 U) z" u

The THP promoter was used to achieve TAL-specific gene disruption because of the known localization of THP to the TAL and early distal tubule as well as continuously high levels of THP production. At the outset of these studies, there was no information on the region of the THP promoter that conferred tissue specificity; however, a recent study using 3 kb of the mouse THP promoter indicated that this region was sufficient to achieve TAL-specific reporter (green fluorescent protein) activity ( 14 ). Serendipitously, the THP promoter fragment used to develop THP-CreTag mice was very similar in location and length to the region used in this previous study. Another choice for a TAL-specific promoter could have been that for the bumetanide-sensitive Na -K -Cl - cotransporter (NKCC2) gene. A small fragment of the mouse NKCC2 promoter has been shown to confer TAL-specific activity in vitro ( 6 ).
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Although no eYFP fluorescence was detected in nonrenal organs and no THP mRNA was expressed outside of the kidney, we did observe substantial CreTag mRNA in testes and very minimal CreTag mRNA in the brain. The significance of CreTag mRNA expression in these nonrenal organs is unclear given the lack of eYFP fluorescence and hence no evidence of Cre-mediated recombination. The substantial signal in the testes is interesting in light of unpublished reports that Cre-expressing mouse lines must be maintained through the maternal line because the paternal line often yields nonspecific recombination in multiple organs. At this point, it seems most reasonable to selectively use female THP-CreTag mice for breeding with mice containing loxP-flanked genes to achieve TAL-specific recombination., {# _7 \% o' s) `) i3 e

Although THP-CreTag mice exhibit high expression of Cre recombinase, presumably due to ongoing activity of the THP promoter fragment, it is conceivable that Cre recombinase activity in TAL from these mice could be augmented even further. Increased dietary salt (1-15 days) or administration of a loop diuretic (4 days) has been shown to increase protein and steady-state mRNA levels of THP in rats ( 13 ). Although this study did not assess whether THP gene transcription was in fact enhanced, if such an effect did occur, then feeding mice that are heterozygous for both THP-CreTag and the loxP-flanked gene of interest a high-salt diet for only 1 day might result in significant increases in Cre activity.
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There is a wide range of potential uses for mice expressing Cre recombinase selectively in the TAL. Obviously, any gene can be engineered to contain loxP sequences flanking DNA crucial to expression of the biologically active protein. In the TAL, targets of interest include 1 ) ion transporters; 2 ) intracellular regulatory or trafficking proteins; 3 ) secreted proteins suspected to function in an autocrine or paracrine manner such as cyclooxygenase-2, endothelin-1, NOS isoforms, epoxygenases, and others; 4 ) cell surface receptors for any number of autocrine, paracrine, or endocrine factors; or 5 ) proteins implicated in disease processes (e.g., polycystin, epidermal growth factor, transforming growth factor-, etc.). The THP-CreTag transgenic mice developed in this study should, using Cre/lox strategies, greatly facilitate our understanding of TAL physiology and pathophysiology.2 f0 f" ~4 O0 c  n
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ACKNOWLEDGMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59047 (to D. E. Kohan).
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