1 Department of Internal
Medicine, The Cre/loxP and Flp/FRT systems mediate site-specific DNA
recombination and are being increasingly utilized to study gene function in vivo. These systems allow targeted gene disruption in a
single cell type in vivo, thereby permitting study of the physiological
and pathophysiological impact of a given gene product derived from a
particular cell type. In the kidney, the Cre/loxP system has been
employed to achieve gene deletion selectively within principal cells of
the collecting duct. Disruption of target genes in the collecting duct,
such as endothelin-1 or polycystic kidney disease-1
(PKD1), could lead to important insights into the
biological roles of these gene products. With selection of the
appropriate renal cell-specific promoters, these recombination systems
could be used to target gene disruption to virtually any renal cell
type. Although transgenic studies utilizing these recombination systems
are promising, they are in their relative infancy and can be time
consuming and expensive and yield unanticipated results. It is anticipated that continued experience with these systems will
produce an important tool for analyzing gene function in renal health
and disease.
Cre recombinase; loxP; aquaporin-2
GENE TARGETING TECHNOLOGY can be a powerful
tool for studying gene function in vivo. Although this technique has
shed light on many developmental biological questions, it has not
achieved the same success in explaining physiological and
pathophysiological processes in mature animals. The reasons for such
relatively poor results are largely twofold. First, it was initially
not possible to control the timing of gene disruption. Originally, gene
targeting typically involved insertion, using homologous recombination
in mouse embryonic stem (ES) cells, of an exogenous DNA fragment into
an exon critical for target gene function, resulting in gene "knockout" (37). Animals derived from these stem cells are
affected by mutant gene dysfunction throughout ontogenesis, often
yielding undesired effects. For example, endothelins-1 and -3 (ET-1 and ET-3) were initially implicated in blood pressure regulation (46, 47);
however, homozygous ET-1 knockout mice die at birth from first
pharyngeal arch malformation (20), and homozygous ET-3 knockout mice
die shortly after birth due to failure to develop a
myenteric plexus (3). In these cases, the biological roles of ET-1 and
ET-3 could not be studied in mature mice. The second major reason why
traditional gene targeting has had limited success is that the targeted
gene is affected in all cell types. Thus, if one wanted to examine the
biological significance of a targeted gene in a particular cell type,
then this would be precluded by the confounding and potentially
injurious effects of gene dysfunction throughout the body. Hence,
the need arose to conditionally regulate gene targeting. This review
will focus on recently developed techniques used to control the
cell site, timing, and type of gene targeting. In addition, the utility
of these new research strategies in studying renal function is discussed.
Cre/loxP and FLP/FRT Recombinase Systems
ABSTRACT
TOP
ABSTRACT
ARTICLE
REFERENCES
ARTICLE
TOP
ABSTRACT
ARTICLE
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Schematic of Cre-mediated recombination of a gene fragment (gene X)
flanked by loxP sites. Gene X is either excised or inverted if loxP
sites are in the same or opposite orientations, respectively.
A major advantage of the Cre-loxP system lies in its relative simplicity. First, no cofactors are required for Cre activity. This is a result of the Cre/loxP complex providing the necessary energy through formation of phosphotyrosine intermediates at the point of strand exchange (1, 13). Second, loxP target sites are small and easily synthesized. Third, there are no apparent external energy requirements. Fourth, Cre is a very stable protein. Finally, and most important, it is easy to generate DNA constructs with any promoter of interest driving Cre expression (see below). This permits controlling the tissue site, and possibly the timing, of Cre expression and resultant gene disruption. It is not surprising, therefore, that this system has been increasingly employed in manipulating eukaryotic genes in vivo.
Utilization of the Cre/loxP system for gene targeting in vivo involves
two lines of mice (Fig. 2). The first mouse
line is generated using ES cell technology. Typically, the target gene is altered, by homologous recombination in ES cells, such that genomic
regions critical for protein activity are flanked by loxP sites
("floxed" gene). Mice derived from these ES cells
should ultimately contain floxed alleles in all cells. These mice
should be phenotypically normal because the loxP sites were inserted into introns where they theoretically do not affect gene function. The
second line of transgenic mice is generated by standard oocyte injection techniques. These mice express Cre under the control of a
transgenic promoter. Mating of the two mouse lines should result in
Cre-mediated gene disruption only in those cells in which the promoter
is active.
|
Clearly, the power and versatility of the Cre/loxP system is largely a
function of promoter activity. Such activity can be regulated by either
1) endogenous cell-specific elements
or 2) exogenously administered
regulatory (inducing) factors. Several promoters have been used to
achieve tissue-specific Cre expression in mice, including
the lck promoter (thymocytes) (12),
the alpha A-crystallin promoter (eye lens) (21), the
alpha-calcium-calmodulin-dependent kinase II promoter (hippocampus and
neocortex) (42), the whey acidic protein promoter (mammary gland) (44),
the aP2 enhancer/promoter (adipose tissue) (2), the aquaporin-2 (AQP2)
promoter (renal collecting duct) (28), and the mouse myogenin promoter
(skeletal muscle) (11). Notably, most of the studies examining Cre
activity only used a reporter system (a floxed STOP
sequence is inserted 5' to a reporter gene; upon Cre-mediated
recombination, the STOP is excised allowing for expression of the
reporter). One group (12) did use the
lck promoter coupled to Cre to disrupt
the DNA polymerase gene in T cells; however, only ~60% of T
cells were affected. Thus, although there is precedent for
tissue-specific Cre expression, these studies must be interpreted
cautiously until it is clearly demonstrated that genes are disrupted
only in the cell type(s) of interest and in the entire population of
targeted cells (see discussion below).
Cre expression may also be temporally regulated using inducible promoters. This has the theoretical advantage of timing gene targeting events to a particular time in the animal's life, thereby avoiding potentially adverse consequences of defective gene function during earlier developmental stages. A few inducible systems have been coupled to Cre expression, including the interferon-responsive Mx1 promoter (19), a tamoxifen-dependent mutated estrogen receptor promoter (6), and the tetracycline-regulated transactivator/tet operator system (39). Although this is an appealing prospect, these techniques have not yet demonstrated tight control of Cre expression in vivo and cannot, therefore, be recommended at this point.
Flp/FRT system. The Flp/FRT recombination system is essentially the eukaryotic homolog of the Cre/loxP system (34). Flp, a 423 amino acid monomeric peptide encoded within the 2-µm plasmid of Saccharomyces cerevisiae, is very similar to Cre in that it requires no cofactors, uses a phosphotyrosine intermediate for energy, and is relatively stable. FRT is also very similar to loxP in that it is composed of three 13-bp repeats surrounding an 8-bp asymmetric spacer region. The asymmetric region dictates whether excision (FRT sites in same orientation) or inversion (FRT sites in inverted orientation) of an intervening DNA sequence occurs after recombination.
Although not as widely used as Cre/loxP, the Flp/FRT system has been shown to cause site-specific DNA recombination in ES cells and transgenic mice (5). Interestingly, despite similar mechanisms of action and DNA recognition sites, the Cre/loxP and Flp/FRT systems do not exhibit significant cross-reactivity (26). The uniqueness of these two recombination systems may allow them to be used in concert to simplify the gene targeting process (26).
Conditional Gene Targeting in the Kidney
This section will discuss the AQP2-CreTag transgenic mice as an example of conditional gene targeting in the kidney (28). Our goal was to examine renal principal cell-specific gene function by targeting gene knockout to this particular cell type. Such mice would be useful for studying the principal cell-specific function of gene products that are widely expressed in the body and/or genes whose germline targeting leads to fetal or early neonatal death. For the reasons discussed earlier, the Cre/loxP recombination system was employed.The first step toward achieving principal cell-specific gene targeting was creating a transgene in which the AQP2 5'-flanking region drove expression of Cre recombinase. The AQP2 water channel gene promoter was chosen, since it was thought to be selectively active in renal principal cells (10, 29). In brief, 14 kb of the AQP2 gene 5'-flanking region, including the transcription initiation site, was linked to a Cre cassette with a eukaryotic translational initiation site, an amino-terminal SV40 nuclear localization signal (to ensure effective nuclear expression; Ref. 12), a carboxy-terminal HSV glycoprotein epitope tag (to facilitate immunodetection; Ref. 17), and an intronless SV40 late region polyadenylation signal (to enhance mRNA expression; Ref. 9). This transgene was termed AQP2-CreTag and is described in detail elsewhere (41).
AQP2-CreTag transgenic mice were generated by injection of the
transgene into the male pronucleus of fertilized single cell embryos,
and the embryos were implanted into pseudopregnant females (15). Three transgenic founder mice were identified by
PCR amplification of the transgene from tail DNA, and, since transgene
expression depends on integration site, each founder was individually
bred as a line of mice for transgene expression analysis. Next, CreTag and AQP2 mRNA expression were determined by RT-PCR of whole organ RNA
derived from each AQP2-Cre transgenic mouse line. Two lines expressed
CreTag mRNA selectively in kidney, testes and vas
deferens. Similarly, AQP2 mRNA was detected only in these
organs. Thus AQP2 promoter activity, as determined by mRNA expression,
appeared to be confined to renal principal cells and the male
reproductive tract. CreTag and AQP2 protein expression were determined
within the kidney and male reproductive system by immunocytochemistry and immunoblotting. As predicted by the mRNA studied, AQP2 and CreTag
were detected in renal principal cells (Fig.
3), epithelial cells within the vas
deferens, and seminiferous tubules. Double-label immunofluorescence of
the kidney demonstrated that renal CreTag expression was limited to
AQP2-expressing collecting duct principal cells. Notably, CreTag was
not found in all renal principal cells (i.e., expression was
variegated), a common finding when many transgene copies are
chromosomally integrated and the transgene includes prokaryotic DNA
(33).
|
It was next important to demonstrate that CreTag was active in vivo.
This was accomplished by breeding the AQP2-CreTag mouse with a reporter
mouse. The reporter mouse contained a loxP-STOP-loxP-lacZ transgene
targeted to the RNA polymerase II gene promoter (RNA PolII
promoter-loxP-STOP-loxP-lacZ; Ref. 30). Doubly
heterozygous offspring of this mating should excise the STOP sequence
in cells expressing CreTag, thereby permitting expression of lacZ (blue cells when stained by X-Gal), whereas cells not
expressing CreTag should remain unstained by X-Gal. As predicted,
mating female AQP2-CreTag mice with male lacZ reporter mice resulted in
X-Gal-stained blue cells in testes, vas deferens, and kidney medulla
(Fig. 4). Interestingly, when male
AQP2-CreTag mice were bred with female lacZ mice, the STOP was excised
from all organs, suggesting that CreTag contained in sperm excised the
STOP shortly after fertilization.
|
In summary, AQP2-CreTag mice express functionally active Cre in renal principal cells and the male reproductive tract. The finding of male reproductive tract AQP2-CreTag transgene expression was unexpected and underscores the difficulty in obtaining tissue-specific promoter activity. Nonetheless, we anticipate that these mice will still be highly useful in analyzing renal principal cell-specific gene function as long as coincident target gene deletion in the male reproductive tract does not have undesirable effects. As will be described below, it is anticipated that these mice will be highly useful in analysis of principal cell-specific gene function as well as in other applications.
Potential Applications of Conditional Gene Targeting for Renal Research
It is evident that genes might be targeted in almost any renal cell type. The ability to achieve cell-specific gene targeting depends upon the promoter driving Cre or Flp expression. Several renal cell-specific promoters have been identified, including those that may selectively target renal collecting duct intercalated cells [anion exchanger (AE1) or B-subunit of the proton ATPase; Refs. 27 and 35], renal collecting duct principal cells (vasopressin V2 receptor and vasopressin-regulated urea transporter; Refs. 22 and 38), thick ascending limb (Tamm-Horsfall protein and NKCC2 Na-K-Cl cotransporter; Refs 16 and 31), thin limbs of Henle's loop (ClC-K1 kidney chloride channel; Ref. 43), proximal tubuleGiven that one could theoretically target virtually every cell type in
the kidney, what genes would be likely candidates for knockout or
knock-in? This is perhaps the most daunting challenge: selecting a gene
of interest knowing that any gene that has been cloned can be targeted.
Obvious candidate genes relevant to renal research include those
encoding 1) transporter proteins;
2) intracellular regulatory or
trafficking proteins; 3) secreted
proteins suspected to function in an autocrine or paracrine manner in
the kidney; 4) cell surface
receptors for any number of autocrine, paracrine, or endocrine factors
affecting renal cell function; or 5)
proteins implicated in disease processes (e.g., polycystin, epidermal
growth factor, transforming growth factor-, etc.). Consider two
examples. ET-1 has been implicated in the regulation of body volume
homeostasis by virtue of its ability to modulate renal vascular tone
and renal cell sodium and water transport (18). The peptide is produced by and binds to multiple renal cell types, each of which could contribute to the effects of ET-1 on renal salt and water excretion (18). The collecting duct is, however, the predominant nephron site of
ET-1 production and binding; hence, we have been interested in the role
of collecting duct-derived ET-1 in the physiological and
pathophysiological regulation of renal fluid reabsorption. Unfortunately, no standard technique (ET-1 antibodies, antagonists, conventional gene targeting) selectively blocks collecting duct-derived ET-1. This problem could, however, potentially be circumvented by
breeding the AQP2-Cre-expressing mouse with a mouse containing a floxed
ET-1 gene, thereby deleting ET-1 production selectively in the
collecting duct. As a second example, no animal model of autosomal
dominant polycystic kidney disease due to PKD1 gene deficiency has been
successfully developed. Conventional knockout of the PKD1 gene results
in mice dying in the perinatal period associated with replacement of
all normal renal parenchyma with cysts (24); hence, these mice cannot
be used to study the progressive renal cyst formation that typically
occurs during adulthood in this disease. One potential way to resolve
this problem is to breed the AQP2-Cre-expressing mouse with a mouse
containing a floxed PKD1 gene. Subsequent breedings should yield a
mouse in which PKD1 gene function is disrupted only in the collecting
duct. This may lead to localized renal cyst formation, thereby
preventing perinatal mortality and permitting analysis of cyst
progression during adulthood. Clearly, the possibilities are virtually limitless.
Potential Limitations of Conditional Gene Targeting
Although Cre- or Flp-mediated gene targeting holds much promise, significant potential limitations exist. There are several concerns with regard to the promoter-cre gene mice. As alluded to above, because of chromosomal integration site and inherent promoter activity, many transgenic animal lines may need to be screened before one with the desired cell-specific expression is identified. In addition, not all targeted cells may produce Cre, i.e., Cre expression is variegated. The reasons for this are multiple (7, 8, 45), but again, many lines of transgenic animals may need to evaluated if Cre activity in all targeted cells is desired (variegated expression may be useful in some circumstances to compare the biological effects of gene disruption in one cell versus a neighboring normal control cell). It is also critically important to document recombination at the floxed gene locus (e.g., mating loxP-expressing mice with lacZ reporter mice). This is particularly important since immunodetection may underestimate the number of cells with functional active Cre recombinase. With regard to mice with floxed genes, it is important to note that these are quite expensive and time consuming to generate. Several commercial organizations charge between $60,000-70,000 US to generate a mouse containing the floxed gene, and the process does not uncommonly take between 5-10 mo. Furthermore, if knockouts are being attempted, then the region of the gene to be flanked by loxP sites must be carefully selected, otherwise truncated or mutated proteins with biological activity may be synthesized. Finally, mice ultimately containing targeted gene disruptions may have phenotypes of uncertain significance. For example, if no unique phenotype is obtained, then does this reflect the biological insignificance of the targeted gene or potentially complex compensatory mechanisms? Also, if the usual system is devised such that the target gene is disrupted during embryogenesis, then unanticipated developmental changes may still occur.Summary
The Cre/loxP and Flp/FRT recombination systems can be used to achieve cell-specific gene targeting in the kidney and other organs. These systems hold much promise for facilitating study of the contribution of genes products derived from specific cell types to physiological and pathophysiological processes. The power of these systems lies in 1) their ability to target any gene; and 2) their ability to target a given gene in any cell type for which a cell type-specific promoter exists. Conditional gene targeting should only be undertaken, however, after careful considerations of the time, expense, and potential complicating factors. This technique, while in its relative infancy, holds much promise as a means to explore heretofore unanswerable questions. ![]() |
ACKNOWLEDGEMENTS |
---|
Portions of this work were funded by the Primary Children's Research Foundation (R. D. Nelson), by National Institutes of Health Grants DK-02132-06 (to R. D. Nelson), DK-52043 (to D. E. Kohan), and HL-56857 (to D. E. Kohan), and by National Cancer Institute Grant 5-P30-CA-42014 (to R. D. Nelson and D. E. Kohan).
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. E. Kohan, Medical Care Center (111), Salt Lake Veterans Affairs Medical Center, 500 Foothill Blvd., Salt Lake City, UT 84148 (E-mail: kohan.donald{at}salt-lake.va.gov).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
1.
Abremski, K.,
and
R. Hoess.
Bacteriophage P1 site-specific recombination.
J. Biol. Chem.
259:
1509-1514,
1984
2.
Barlow, C.,
M. Schroeder,
J. Lekstron-Himes,
H. Kylefjord,
C.-X. Deng,
A. Wynshaw-Boris,
B. Speigelman,
and
K. Xanthopoulos.
Targeted expression of Cre recombinase to adipose tissue of transgenic mice directs adipose-specific excision of loxP-flanked gene segments.
Nucleic Acids Res.
25:
2543-2545,
1997
3.
Baynash, A.,
K. Hosoda,
A. Giald,
J. Richardson,
N. Emoto,
R. Hammer,
and
M. Yanagisawa.
Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.
Cell
79:
1277-1285,
1994[Medline].
4.
Ding, Y.,
R. Davisson,
D. Hardy,
L.-J. Zhu,
D. Merrill,
J. Catterall,
and
C. Sigmund.
The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice.
J. Biol. Chem.
272:
28142-28148,
1997
5.
Dymecki, S.
Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice.
Proc. Natl. Acad. Sci. USA
93:
6191-6196,
1996
6.
Feil, R.,
J. Brocard,
B. Mascrez,
M. LeMeur,
D. Metzger,
and
P. Chambon.
Ligand-activated site-specific recombination in mice.
Proc. Natl. Acad. Sci. USA
93:
10887-10890,
1996
7.
Felsenfeld, G.,
J. Boyes,
J. Chung,
D. Clark,
and
V. Studitsky.
Chromatin structure and gene expression.
Proc. Natl. Acad. Sci. USA
93:
9384-9388,
1996
8.
Festenstein, R.,
M. Tolaini,
P. Corbella,
C. Mamalaki,
J. Parrington,
M. Fox,
A. Miliou,
M. Jones,
and
D. Kioussis.
Locus control region function and heterochromatin-induced position effect variegation.
Science
271:
1123-1125,
1996[Abstract].
9.
Fitzgerald, M.,
and
T. Shenk.
The sequence 5'-AAUAAA-3' forms parts of the recognition site for polyadenylation of late SV40 mRNAs.
Cell
24:
251-260,
1981[Medline].
10.
Fushimi, K.,
S. Uchida,
Y. Hara,
Y. Hirata,
F. Marumo,
and
S. Sasaki.
Cloning and expression of apical membrane water channel of rat kidney collecting tubule.
Nature
361:
549-552,
1993[Medline].
11.
Grieshammer, U.,
M. Lewandoski,
D. Prevette,
R. Oppenheim,
and
G. Martin.
Muscle-specific cell ablation conditional upon Cre-mediated DNA recombination in transgenic mice leads to massive spinal and cranial motoneuron loss.
Dev. Biol.
197:
234-247,
1998[Medline].
12.
Gu, H.,
J. Marth,
P. Orban,
H. Mossmann,
and
K. Rajewsky.
Deletion of a DNA polymerase gene segment in T cells using cell type-specific gene targeting.
Science
265:
103-106,
1994[Medline].
13.
Guo, F.,
D. Gopaul,
and
G. Van Dyne.
Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse.
Nature
389:
40-46,
1997[Medline].
14.
Hoess, R.,
A. Wierzbicki,
and
K. Abremski.
The role of the spacer region in P1 site-specific recombination.
Nucleic Acids Res.
14:
2287-2300,
1986[Abstract].
15.
Hogan, B.,
F. Costantini,
and
E. Lacy.
Manipulating the Mouse Embryo. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1986.
16.
Igarashi, P.,
D. Whyte,
K. Li,
and
G. Nagami.
Cloning and kidney cell-specific activity of the promoter of the murine Na-K-Cl cotransporter gene.
J. Biol. Chem.
271:
9666-9674,
1996
17.
Isola, V. J.,
R. J. Eisenberg,
G. R. Siebert,
C. J. Heilman,
W. C. Wilcox,
and
G. H. Cohen.
Fine mapping of antigenic site II of herpes simplex virus glycoprotein D.
J. Virol.
63:
2325-2334,
1989[Medline].
18.
Kohan, D.
Endothelins in the normal and diseased kidney.
Am. J. Kidney Dis.
29:
2-26,
1997[Medline].
19.
Kühn, R.,
F. Schwenk,
M. Aguet,
and
K. Rajewsky.
Inducible gene targeting in mice.
Science
269:
1427-1429,
1995[Medline].
20.
Kurihara, Y.,
H. Kurihara,
H. Suzuki,
T. Kodama,
K. Maemura,
R. Nagai,
H. Oda,
T. Kuwaki,
W. Cao,
N. Kamada,
K. Hishage,
Y. Ouchi,
S. Azuma,
Y. Toyada,
T. Ishikawa,
M. Kumada,
and
Y. Yazaki.
Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1.
Nature
368:
703-710,
1994[Medline].
21.
Lakso, M.,
B. Sauer,
J. B. Mosinger,
E. Lee,
R. Manning,
S.-H. Yu,
K. Mulder,
and
H. Westphal.
Targeted oncogene activation by site-specific recombination in transgenic mice.
Proc. Natl. Acad. Sci. USA
89:
6232-6236,
1992[Abstract].
22.
Lolait, S.,
A.-M. O'Carroll,
O. McBride,
M. Konig,
A. Morel,
and
M. Brownstein.
Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus.
Nature
357:
336-339,
1992[Medline].
23.
Loya, F.,
Y. Yang,
H. Lin,
E. Goldwasser,
and
M. Albitar.
Transgenic mice carrying the erythropoietin gene promoter linked to lacZ express the reporter in proximal convoluted tubule cells after hypoxia.
Blood
84:
1831-1836,
1994
24.
Lu, W.,
B. Peissel,
H. Babakhanlou,
A. Pavlova,
L. Geng,
X. Fan,
C. Larson,
G. Brent,
and
J. Zhou.
Perinatal lethality with kidney and pancreas defects in mice with a targeted Pkd1 mutation.
Nat. Genet.
17:
179-181,
1997[Medline].
25.
Magagnin, S.,
A. Werner,
D. Markovich,
V. Sorribas,
G. Stange,
J. Biber,
and
H. Murer.
Expression cloning of human and rat renal cortex Na/Pi cotransport.
Proc. Natl. Acad. Sci. USA
90:
5979-5983,
1993[Abstract].
26.
Meyers, E.,
M. Lewandoski,
and
G. Martin.
An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination.
Nat. Genet.
18:
136-141,
1998[Medline].
27.
Nelson, R.,
X. Guo,
K. Masood,
D. Brown,
M. Kalkbrenner,
and
S. Gluck.
Selectively amplified expression of an isoform of the vacuolar H(+)-ATPase 56-kilodalton subunit in renal intercalated cells.
Proc. Natl. Acad. Sci. USA
89:
3541-3545,
1992[Abstract].
28.
Nelson, R.,
P. Stricklett,
D. Ausiello,
D. Brown,
and
D. Kohan.
Expression of a Cre recombinase transgene by the aquaporin-2 promoter in kidney and male reproductive system of transgenic mice.
Am. J. Physiol.
275 (Cell Physiol. 44):
C216-C226,
1998
29.
Nielsen, S.,
S. R. DiGiovanni,
E. I. Christensen,
M. A. Knepper,
and
H. W. Harris.
Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.
Proc. Natl. Acad. Sci. USA
90:
11663-11667,
1993[Abstract].
30.
O'Gorman, S.,
N. Dagenais,
M. Qian,
and
Y. Marchuk.
Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells.
Proc. Natl. Acad. Sci. USA
94:
14602-14607,
1997
31.
Pennica, D.,
W. Kohr,
W.-J. Kuang,
D. Glaister,
B. Aggarwal,
E. Chen,
and
D. Goeddel.
Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein.
Science
236:
83-88,
1987[Medline].
32.
Pietromonaco, S.,
D. Kerjaschki,
S. Binder,
R. Ullrich,
and
M. Farquhar.
Molecular cloning of a cDNA encoding a major pathogenic domain of the Heymann nephritis antigen gp330.
Proc. Natl. Acad. Sci. USA
87:
1811-1815,
1990[Abstract].
33.
Robertson, G.,
D. Garrick,
W. Wu,
M. Kearns,
D. Martin,
and
E. Whitelaw.
Position-dependent variegation of globin transgene expression in mice.
Proc. Natl. Acad. Sci. USA
92:
5371-5375,
1995[Abstract].
34.
Sadowski, P.
The Flp recombinase of the 2-µm plasmid of Saccharomyces cerevisiae.
Prog. Nucleic Acid Res. Mol. Biol.
51:
53-91,
1995[Medline].
35.
Sahr, K.,
W. Taylor,
B. Daniels,
H. Rubin,
and
P. Jarolim.
The structure and organization of the human erythroid anion exchanger (AE1) gene.
Genomics
24:
491-501,
1994[Medline].
36.
Schaffner, S.,
D.-F. Wan,
G. Habib,
E. Banez,
C. Ou,
S. Rajagopalan,
E. Aguilar-Cordova,
R. Lebovitz,
R. Overbeek,
and
M. Lieberman.
Targeting of the rasT24 oncogene to the proximal convoluted tubules in transgenic mice results in hyperplasia and polycystic kidneys.
Am. J. Pathol.
142:
1051-1060,
1993[Abstract].
37.
Sedivy, J.,
and
A. Joyner.
Gene Targeting. New York: Freeman, 1992.
38.
Shayakul, C.,
A. Steel,
and
M. Hediger.
Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts.
J. Clin. Invest.
98:
2580-2587,
1996
39.
St.-Onge, L.,
P. Furth,
and
P. Gruss.
Temporal control of the Cre recombinase in transgenic mice by a tetracycline responsive promoter.
Nucleic Acids Res.
24:
3875-3877,
1996
40.
Sternberg, N.,
and
D. Hamilton.
Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites.
J. Mol. Biol.
150:
467-486,
1981[Medline].
41.
Stricklett, P.,
R. Nelson,
and
D. Kohan.
Site-specific recombination using an epitope-tagged bacteriophage P1 Cre recombinase.
Gene
215:
415-423,
1998[Medline].
42.
Tsien, J.,
P. Huerta,
and
S. Tonegawa.
The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory.
Cell
87:
1327-1338,
1996[Medline].
43.
Uchida, S.,
T. Rai,
H. Yatsushige,
Y. Matsumura,
M. Kawasaki,
S. Sasaki,
and
F. Marumo.
Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter.
Am. J. Physiol.
274 (Renal Physiol. 43):
F602-F610,
1998
44.
Wagner, K.-U.,
R. Wall,
L. St.-Onge,
P. Gruss,
A. Wynshaw-Boris,
L. Garrett,
M. Li,
P. Furth,
and
L. Hennighausen.
Cre-mediated gene deletion in the mammary gland.
Nucleic Acids Res.
25:
4323-4330,
1997
45.
Wilson, C.,
H. Bellen,
and
W. Gehring.
Position effects on eukaryotic gene expression.
Annu. Rev. Cell Biol.
6:
679-714,
1990.
46.
Yanagisawa, M.,
A. Inoue,
T. Ishikawa,
Y. Kasuya,
S. Kimura,
S.-I. Kumagaye,
K. Nakajima,
T. Watanabe,
S. Sakakibara,
K. Goto,
and
T. Masaki.
Primary structure, synthesis, and biological activity of rat endothelin, and endothelium-derived vasoconstrictor peptide.
Proc. Natl. Acad. Sci. USA
85:
6964-6967,
1988[Abstract].
47.
Yanagisawa, M.,
H. Kurihara,
S. Kimura,
Y. Tomobe,
M. Kobayashi,
Y. Mitsui,
Y. Yazaki,
K. Goto,
and
T. Masaki.
A novel potent vasoconstrictor peptide produced by vascular endothelial cells.
Nature
332:
411-415,
1988[Medline].