Conditional Gene Targeting

Alexander Gawlik and Susan E. Quaggin Physiology 19:245-252, 2004. doi:10.1152/physiol.00009.2004 You might find this additional information useful... This article cites 44 articles, 24 of which you can access free at: http://physiologyonline.physiology.org/cgi/content/full/19/5/245#BIBL Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl on the following topics: Biochemistry .. Recombinases Oncology .. Gene Function Oncology .. Gene Silencing Oncology .. Gene Therapy Pharmacology .. Antibiotics Veterinary Science .. Mammalia Updated information and services including high-resolution figures, can be found at: http://physiologyonline.physiology.org/cgi/content/full/19/5/245 Additional material and information about Physiology can be found at: http://www.the-aps.org/publications/physiol

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PHYSIOLOGY 19: 245–252, 2004; 10.1152/physiol.00009.2004

Deciphering the Renal Code: Advances in Conditional Gene Targeting Several powerful new techniques can examine gene function in mammals. Recombinase systems and kidney-specific promoters enable gene knockout and overexpression. Genetic systems induced on administration or removal of

Alexander Gawlik1,2 and Susan E. Quaggin1,3 1

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto M5G 1X5; 2 Deptartment of Medicine II, University Hospital Aachen, 52070 Aachen, Germany; and 3 Division of Nephrology, Department of Medicine, St. Michael’s Hospital, University of Toronto, Toronto M5B 1W8, Canada [email protected]

antibiotics or hormones permit control of gene expression. Gene silencing using short interfering RNA expression systems should accelerate loss-of-function studies. Thorough characterization of animals that have undergone conditional gene targeting has already provided insights into renal development and diseases. Here we discuss the advantages and pitfalls of currently available genetargeting systems. Downloaded from physiologyonline.physiology.org on September 27, 2007

Complete mapping of the genetic code in several mammalian organisms has led to the need for functional characterization of a vast number of different gene sequences. Since 1985, investigators using mouse embryonic stem (ES) cells have been able to modify a gene locus by homologous recombination. Using this technique, loss- and gain-of-function studies for specific target genes can be performed in vivo. Because of the remarkable similarities between murine and human gene and cellular functions, the mouse is an excellent model system to study human biology and disease. This has established gene targeting in mice as a widely used and powerful tool for gene function studies. Although conventional targeting within the germline has provided great insight into the biological functions of many genes, several disadvantages exist. Disruption of gene function in ES cells may result in embryonic or perinatal lethality, preventing the functional characterization of the gene in organs such as the kidney that develop relatively late in fetal life. Additionally, many genes are expressed in multiple cell types, and the resulting knockout phenotypes can be complex and difficult or impossible to dissect. The ability to limit gene targeting to a specific cell type overcomes some of these problems, and the temporal control of gene expression permits more precise dissection of a gene’s function. Moreover, if a gene’s function is dependent on tight regulation of its expression, knockout and overexpression studies represent two extremes. In this case, generation of mice with intermediate levels of gene expression is advantageous. Therefore, an ideal tool to manipulate gene function would permit tight control over the spatial and temporal levels of expression while being cost effective and efficient. New developments in genetic engineering should allow realization of many of these criteria.

Kidney-Specific Promoters Several kidney-specific promoters have now been characterized, making it possible to drive gene expression under spatial control. Within the tubule, proximal cells can be targeted by using a chimeric construct containing a 1,542-bp fragment of the 5 flanking region of the kidney androgen-regulated promoter (KAP) gene and the remainder of the human angiotensinogen (hAGT) structural gene starting with the last 36 nucleotides of exon II through the poly(A) addition site (25) or 346 bp of the -glutamyl transpeptidase type II promoter (36). Three kilobases of the Tamm horsfall protein (THP) promoter directs expression to the thick ascending limb of the loop of Henle (TALH) and early distal convoluted tubules (44). In contrast, 1.34 kb of the kidney-specific (Ksp) cadherin promoter directs expression to the TALH and collecting ducts of the adult nephron and weakly to other tubular cell types and to the ureteric bud, Wolffian duct, Mullerian duct, and developing tubules in the mesonephros and metanephros (17). A 324-bp fragment limits expression to tubular epithelia of the developing kidney and genitourinary tract (37). The HoxB7 promoter marks the cells within the ureteric bud and its derivatives (40). Within the glomerulus, substantial progress has been made in podocyte-specific gene targeting. A 1.25-kb fragment of the human nephrin (NPHS1) promoter and 8.3-kb, 5.4-kb, 4.125-kb, and 1.25-kb fragments of the murine NPHS1 promoter direct expression to podocytes (13, 29, 42). A 2.5-kb fragment of the promoter from a second gene, podocin (NPHS2), also directs expression to podocytes (31). Despite the growing list of kidney-specific promoters, certain cell types remain untouchable in vivo, including mesangial cells and subpopulations

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Use of Site-Specific Recombinases in the Kidney In addition to overexpression studies, investigators often wish to knock down or knock out the function of a specific protein within the kidney. Although expression of a dominant-negative protein may be relevant to human disease or the pathway under study, deletion of a gene within the genome of a specific cell type is also valuable to study gene function. To facilitate the generation of kidneyspecific knockouts, site-specific recombinase systems have been used. Of the recombinase systems available, the CreloxP system has been most widely used in mice. Cre recombinase is a 38-kDa protein that recognizes a 34-bp DNA target called locus of X-over of P1 (loxP) and was discovered in the bacteriophage P1 (24, 34). This minimal target sequence site is unlikely to occur randomly in the mouse genome and is small enough to be “neutral” when integrated into chromosomal DNA. If two loxP sites are located on the same DNA molecule, Cre causes inversion or excision of the intervening DNA segment depending 246

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on their respective orientation. Two transgenic mouse lines are required to facilitate tissue-specific knockouts (FIGURE 1). The first mouse line expresses Cre recombinase under the control of a tissue-specific promoter. The second carries loxP sites around the gene of interest (“floxed gene”) After intercrossing, the gene of interest will be removed selectively from cells expressing Cre recombinase. One or both copies of the gene can be targeted (hetero- or homozygosity of the floxed gene, respectively), permitting a crude examination of dosage sensitivity for a specific gene. The Cre-loxP system can also be used to activate the expression of genes by excision of a “floxed STOP codon” placed between a highly active promoter and the gene of interest. The advantage of this system compared with the standard approach using a cell-specific promoter to directly drive expression of a gene is that it permits robust and reproducible expression of the gene under a wellcharacterized potent promoter, such as the promoter from the chicken
-actin gene. Integration of the transgene into an active part of the genome can be rapidly determined by using a reporter gene such as lacZ or the enhanced green fluorescent protein (EGFP). In a similar manner, activating reporter genes such as EGFP makes cell lineage-tracing studies possible in vivo. Cells can be tagged at specific time points and their fate followed during development or in disease (32). To date, four different kidney-specific Cre mouse lines have been characterized. Under the control of the 4.125-kb fragment of the murine NPHS1 (13) or 2.5-kb fragment of the human NPHS2 5 flanking region (30), Cre-mediated site-specific recombination occurs specifically within podocytes in vivo (FIGURE 1). Under the control of a 1.34-kb fragment of the Ksp cadherin promoter (38), Cre-mediated site-specific recombination occurs within tubular cells. Additionally, the ureteric bud and its derivatives can be targeted from 9.5 days of embryonic life onward by using a mouse line that expresses Cre recombinase under the control of 1.3 kb of the HoxB7 enhancer/promoter (43). Mx1 Cre mice express Cre recombinase under the control of the IFN-inducible Mx1 promoter, limiting expression to vascular endothelium, liver, spleen, and a subset of stromal cells in uterus, duodenum, colon, and aorta. In the kidney, transgene expression is limited to all glomerular cells and a subset of tubules, excluding proximal tubules and distal convoluted tubules (35). The Cre-loxP system has been used to study the role of vascular endothelial growth factor (VEGF) in renal disease. Mice that lack either one or two alleles of VEGF-A specifically from their podocytes were created. By 2.5 wk of age mice with deletion of one allele of the VEGF-A gene developed protein-

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of metanephric mesenchymal cells and their derivatives. Recently, a 2.5-kb promoter fragment from the 5 flanking region of the megsin gene was described that directs gene expression that is largely restricted to the mesangial cell lineage within the glomerulus in vitro (18, 20). Mesangial cell culture studies have shown that this promoter is functional in vitro (19). Using the published 5 flanking fragment that is functional in vitro, we generated a megsin-lacZ transgene but were unable to detect any transgene expression within mesangial cells in vivo in 20 individual transgenic founder lines (M. Alexander and S. Quaggin, unpublished observations). Because a number of genes have been identified that are highly expressed in metanephric mesenchymal cells, it should be possible to generate cyclization recombination (Cre) recombinase lines that express in these cell types, if not through isolated promoters, then through genomic knockin approaches. Using these promoters, investigators are able to determine whether overexpression of their candidate “disease gene” or a dominant-negative version of their protein (one that interferes with the function of the wild-type protein) leads to kidney disease. However, interpretation of the biological relevance of these models must be done carefully. In particular, it is difficult to control the level of expression tightly with this approach and expression of supraphysiological levels of a gene may not be biologically relevant. Interpretation of experimental results using cell-specific transgene approaches must include these considerations.

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uria and endotheliosis, the renal lesion seen in preeclampsia. Deletion of both alleles in podocytes led to perinatal lethality and failure to develop a filtration barrier due to defects in endothelial cell migration, differentiation, and function. These results emphasize the critical role that VEGF-A signaling plays in the establishment and maintenance of the glomerular filtration barrier (11, 12). As the number of mouse lines with different floxed genes increases exponentially in number, it will be possible to determine the role of these genes in podocytes or tubular epithelial cells simply by crossing two mouse strains and analyzing the offspring. In this regard, numerous mouse phenotyping facilities have emerged that permit high-resolution imaging and physiological analyses (e.g., http://www.cmhd.ca). Furthermore, a database has been established by Nagy and colleagues (http://www.mshri.on.ca/ nagy) that describes the characteristics and contacts for many of the transgenic conditional mouse lines currently available. In addition to the Cre-loxP system, two other recombinase systems have been used successfully

No knockout in nonkidney cells

LoxP

FIGURE 1. Cre-loxP system in the kidney Two transgenic mouse lines are required to achieve a knockout of a gene of interest exclusively in the kidney: one carries loxP sites around a critical portion of or around the entire gene of interest; the other expresses Cre recombinase under the control of a kidney-specific promoter. After intercrossing, Cre recombinase will excise the floxed gene of interest only in kidney cells. All other cells do not express Cre recombinase.

in vivo: the Flp-FRT system from the budding yeast Saccharomyces cerevisiae (9) and -integrase (1). Flp recombinase recognizes a 34-bp consensus sequence known as FRT and induces recombination between two of these sites. The activities of Cre and Flp have different temperature sensitivities in vitro. Cre was shown to be active over a wider range of temperatures than Flp, with a maximal performance at 42°C, and therefore offered a theoretical advantage for use in in vivo systems (4). To improve the activity of Flp, a Flp mutant was developed by introduction of four amino acid changes (Flpe) (4). Flpe has activity in ES cell cultures equivalent to that of Cre and thus should be just as useful for in vivo experiments. Combining both Flp and Cre systems may allow an investigator to target cell lineages that would otherwise be inaccessible. Phage integrases also mediate recombination between short sequences of DNA, the phage attachment site “attP,” and a short sequence of bacterial DNA, the bacterial attachment site “attB.” They are categorized as tyrosine or serine integrases, based on their mode of catalysis. The

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Kidney-specific promoter

Kidney-specific promoter Cre recombinase

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has been reported with other tissue-specific promoter-driven Cre systems is that the promoter itself may “mop up” cell-specific transcription factors and alter the phenotype. This is an important consideration that can be overcome by thorough examination of control mice. On occasion, transmission of both the Cre and floxed transgenes through the same germline leads to ubiquitous deletion, i.e., the cell-specific promoter is active within the germ cell, leading to widespread excision. This is a major concern if there is a haploinsufficient phenotype associated with the floxed allele (e.g., VEGF) (5). From this discussion, it is clear that full characterization of the gene excision in individual Cre lines must be performed. Two excellent reporter mouse strains exist to test cell-specific Cre-mediated excision, known as the Z/AP and Z/EG lines (27, 33). In our experience, the Z/EG line that expresses the EGFP following excision of a floxed STOP allele permits the highest resolution of cell-specific expression when examined using a commercially available anti-GFP antibody (13). Despite the potential pitfalls associated with recombinase systems, as the availability and variety of Cre lines increase, investigators should be able to find lines that suit their experimental needs. In addition, an optimist might consider that mosaic excision or excision after onset of expression of a gene may be desirable and may reveal phenotypes or gene functions that would otherwise be masked by a more complete knockout.

Adding Temporal Control The dissection of the role of specific genes in disease requires temporal control of gene expression in addition to tissue specificity. To achieve this goal, tetracycline-sensitive systems have successfully been employed in the kidney (FIGURE 2). Tetracycline binds to the tetracycline transactivator tTA or “reverse” rtTA. These complexes repress or activate the expression of the gene of interest by binding to the Tet operator (tetO). Shigehara et al. (39) recently produced a transgenic line using the NPHS2 gene promoter to restrict expression of the rtTA cassette to podocytes; they bred these mice with a reporter mouse line that contains the CMV minimal promoter and tetO promoter elements together with lacZ. Administration of tetracycline to the drinking water initiated the expression of the lacZ reporter gene selectively in podocytes. Replacing lacZ with Cre recombinase would enable researchers to benefit from the pool of mouse strains with floxed genes of interest by generating triply transgenic mice that carry the podocyte-specific rtTA, tetracycline-sensitive Cre recombinase transgenes, and the floxed line of interest.

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Streptomyces phage-derived C31, a member of the serine integrase family, has been shown to work efficiently in mammalian cells without any requirement for host cofactors (1). The ability of phage integrases to unidirectionally and irreversibly integrate an external DNA sequence into the genome makes them useful for genetic engineering in ES cells (1). Once an att site is inserted into a specific chromosomal locus by homologous recombination, any gene of interest may theoretically be integrated into that site at relatively high efficiency. With this strategy, it is possible to insert genes of interest into the same genomic locus so that their effect can be studied and compared in the same context. The biology and applications of phage integrases have recently been reviewed elsewhere (15). Despite all of the promising results and insights that recombinase systems have provided, there are several caveats that need to be considered. The degree and timing of Cre-mediated excision during embryonic development is critical, because incomplete excision (<100% of targeted cells) might lead to unexpected results, particularly if the gene of interest is a secreted molecule. The presence of residual wild-type cells may produce a different phenotype than that produced by the complete absence of wild-type cells. Although Cre-mediated excision of a floxed reporter allele may be 100%, this can vary from locus to locus. Efficiency of recombination drops with the distance between loxP sites. Although it is not always possible to determine genomic excision for each floxed gene in every cell, additional steps such as laser capture microscopy (7) of the cell of interest could be performed to ensure cell-specific and complete excision. The exact stage of Cre-mediated excision during embryonic development of the tissue or cell of interest is also important, and if a gene is expressed before the time of excision, a lesssevere phenotype may ensue. The stability of the protein and/or gene products may determine this. Although largely theoretical, endogenous pseudoloxP sites that occur naturally in the mammalian genome have been reported; whether any of these sites are associated with genes expressed by renal cells and if recombination of these sites could alter the phenotype of the kidney is not known. Additionally, using the Cre-loxP system to overexpress genes calls for highly active promoters driving expression ubiquitously. The CMV-enhanced chicken
-actin promoter (pCAGGS) has been most widely used in this context. Evidence is accumulating that shows mosaic transgene expression under this promoter upon cellular differentiation, emphasizing the urgent need for the development of reliable ubiquitous promoters. Another theoretical concern in the kidney that

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Although widely used in vivo, a disTriple-transgenic mouse advantage of the tetracycline system is that to knock out a gene of interest in a temporal manner requires generation FIGURE 2. rtTA-tetO system in the kidney of triple-transgenic offspring. Another To achieve inducible knockout of a gene of technique that overinterest exclusively in the kidney, triple transcomes this problem and genic mice are generated. The reverse tet repressor (rtTR) is expressed under the control can achieve inducible rtTR Kidney-specific promoter of a kidney-specific promoter. Only in the presactivity of site-specific ence of rtTR and delivery of tetracycline is the Tet Tet recombinases is the Tet-operator promoter activated and is Cre Tet recombinase expressed, which excises the gene development of ligandof interest. regulated forms of Cre and Flpe by fusing a mutant estrogen receptor (ER) rtTR ligand-binding domain to the COOH Tet Tet Cre terminal of the enzymes. These fusion Tet-operator promoter recombinase proteins are induced by application of the synthetic estrogen antagonist 4OH tamoxifen but are insensitive to endogenous
LoxP Gene of interest LoxP estradiol. Researchers can choose between three different mutant estrogen receptors: mouse ERTM Excision of (8), human ERT (28), and human ERT2 (14). CreERT2 gene of interest is ~10-fold more sensitive than CreERT (21). By LoxP Gene of interest placing CreERT under the control of a kidney-specific promoter, investigators should be able to generate a simpler system to knock out genes in the LoxP through IFN. In 2001, Elbashir kidney in a temporally controlled manner. and colleagues (10) were able Although clearly this system would be beneficial, it to show that transfection of chemically synthesized has not yet been achieved in the kidney. In our own siRNAs into mammalian cells effectively silenced lab we generated 12 independent CreERT2 lines the expression of a reporter gene in a sequenceunder control of the nephrin promoter (S. E. specific manner. These findings have led to wideQuaggin, unpublished observations) but were spread application of RNA interference in mamnever able to demonstrate excision. malian species. Transfection of chemically synthesized siRNA RNA Interference into cells is of a transient nature. To overcome this problem, techniques have been developed using Creating knockout animals by using homologous DNA vectors expressing substrates that can be conrecombination in ES cells is still costly and time verted into siRNAs in vivo. Expression of these subconsuming. The development of new techniques strates is usually driven by promoters of genes tranhas made it possible to silence gene expression by scribed by RNA polymerase II or III, which produce using short pieces of RNA. RNA interference is a RNA from a DNA template. To generate dsRNA, highly conserved mechanism throughout transfection with two vectors coding for the sense evolution and can be found in plants as well as and antisense strands is necessary. Meanwhile, it humans. RNA interference is the process of has been shown that Dicer can process small hairsequence-specific, posttranscriptional gene pin RNA (shRNA) structures, resulting in the genersilencing initiated by double-stranded RNA ation of micro RNAs (miRNAs) (FIGURE 3). By (dsRNA) that is homologous in sequence to the inhibiting translation, miRNA can effectively silenced gene. The presence of dsRNA in the silence gene expression, making it possible to tarcytoplasm activates Dicer enzymes that cleave the get genes by using only one vector (3). These sysRNA into short pieces (2). The mediators of tems can now be used to generate transgenic anisequence-specific messenger RNA degradation are mals that silence gene expression stably. 21- and 22-nt short interfering RNAs (siRNAs) Two independent groups (6, 23) demonstrated generated by cleavage from longer dsRNAs (10). successful genomic integration and germline These siRNAs are incorporated into a multiprotransmission of a plasmid expressing siRNA against tein RNA-inducing silencing complex (RISC). The Ras-gap or Neil-1. Individual ES cell lines were genantisense strand guides RISC to its homologous erated that showed varying levels of silencing. Of target mRNA, resulting in cleavage. Unfortunately, note, mice derived through germline transmission in mammals the introduction of long dsRNAs leads showed the same level of reduction in the targeted to a nonspecific inhibitory response mediated

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Short hairpin RNA

Dicer

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FIGURE 3. Principles of shRNA and miRNA

miRNP

Self-complementary short pieces of RNA forming hairpin-like structures (shRNA) activate cytoplasmic Dicer enzymes that cleave the RNA into short pieces (miRNA). miRNAs are incorporated into a miRNA-protein-complex (miRNP). The antisense strand guides miRNP to its homologous target mRNA, resulting in inhibition of translation.

miRNP

mRNA

Translation inhibition

gene as the ES cell line from which they were established. This makes it possible to screen ES cell clones for the desired reduction level ( 100%), permitting the rapid generation of an “allelic series”

831 bp

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FIGURE 4. Design of Cre-loxP shRNA vector Sense RNAs (no repressive effect)

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To achieve shRNA expression only in the presence of Cre recombinase, a construct driven by the RNA polymerase III U6 promoter is used (22). Two loxP sites separated by an 813-bp fragment are placed between sense and antisense regions of shRNA, including 6 thymines serving shRNAs as a stop signal for RNA polymerase III. In the presence of Cre recombinase, the 813-bp fragment is excised and functional shRNAs with a 34-nt hairpin loop (loxP sequence) are transcribed.

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TTTTTT

STOP Sense LoxP Antisense

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(i.e., one can look at the phenotype in several lines and determine the effect of more or less expression of a gene on its function). However, the ability to screen expression levels in ES cells before generation of the mouse demands that the gene be expressed in ES cells. Using pronuclear injection, Hasuwa et al. (16) accomplished germline transmission of RNA polymerase III H1 promoter-driven shRNA expression constructs, silencing EGFP expression in EGFP transgenic mice to 18, 4, and 24% of the control level. Gene silencing by RNA interference exclusively in the kidney requires the addition of a recombinase system to the siRNA or shRNA plasmid. This has recently been accomplished in vitro. Kasim et al. (22) demonstrated that shRNA expression can be activated using the Cre-loxP system. To achieve this result, a construct driven by the RNA polymerase III U6 promoter was used (FIGURE 4). Two loxP sites separated by a 813-bp fragment were placed between sense and antisense regions of shRNA. In the absence of Cre recombinase, only sense RNAs without any suppressive effect are transcribed. In the presence of Cre recombinase, recombination occurs between the two loxP sites and the inserted fragment is excised. As a result, only the 34-bp loxP site separates the sense and antisense sequences, and functional shRNAs with a 34-nt hairpin loop (loxP sequence) are transcribed. Coelectroporation of a loxP-containing shRNA construct with GFP-hybridizing sequences together with a Cre expression vector directly into GFPexpressing muscle of a transgenic mouse led to a

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A. Gawlik is supported by grants from the University Hospital in Aachen; S. E. Quaggin holds a Canada Research Chair Tier II and is the recipient of a Premier’s Research Award of Excellence of Ontario. This work is funded by a Canadian Institutes of Health Research grant MOP-62931 to S. E. Quaggin.

Note added in proof While this article was in press, a new kidney-specific Cre line has become available; the mouse glucose transporter sglt2 5 region limits the expression of Cre-recombinase to the proximal tubule. See Rubera I et al., Specific Cre/Lox recombination in the mouse proximal tubule. Am J Soc Nephrol 15, 2050–2056, 2004.

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“knockdown” of GFP expression in vivo. Similarly, injection of “naked” siRNAs alone into the tail vein of GFP-expressing transgenic mice led to reduction in GFP expression (26). Van de Wetering et al. (41) generated an inducible version of this system by replacing 19 bp directly upstream of the transcription start site of the RNA polymerase III H1 promoter with a binding site for the tet repressor protein. In the absence of tetracycline, the tet repressor binds to this site and prevents transcription of the shRNA construct. Addition of tetracycline permits full H1 promoter activity and production of the siRNAs. It should now be possible to adapt these systems to in vivo models that will allow spatial and temporal control over shRNA expression, permitting rapid and highthroughput analysis of gene function in a timely and cost-effective manner. In summary, new developments in genetic engineering have led to significant improvements in conditional gene targeting in the kidney. Investigators are now able to drive the expression of specific genes in specific cell types under temporal control. As the number of cell-specific promoters and Cre lines in the kidneys increases, researchers will be able to target virtually any gene of interest within the kidney. In turn, this should lead to great advances in our knowledge about renal physiology, development, and diseases. T

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