Kidneys Of Transgenic Mice Research Articles

Silica increases cytosolic calcium and causes cell injury in renal cell lines.

The purpose of this study was to clarify the effect of silica-induced cytosolic free calcium mobilization and cell injury in immortalized cell lines from transgenic mice kidney harboring SV40 T-antigen gene. The proximal convoluted tubule (S1)- and the inner medullary collecting tubule (IMCT)-originated cell lines were used. Cytosolic free calcium concentration ([Ca2+]i) was measured employing Fura-2 fluorescence and cell injury was evaluated by a vital dye exclusion procedure. Silica increased [Ca2+]i in a concentration-dependent manner in S1 (60 micrograms/ml-600 micrograms/ml) and IMCT (6 micrograms/ml-600 micrograms/ml). Silica caused a biphasic increase in [Ca2+]i which was composed of an initial rapid rise and following sustained phase. Ca2+ removal from the medium resulted in abolishment of initial and sustained phase of silica (600 micrograms/ml)-induced [Ca2+]i in both cell lines. Silica-induced cell injury was increased in a dose-dependent manner. This silica-induced cell injury was attenuated by the pretreatment with EGTA (100 microM) and nifedipine (1 microM). Cellular ATP content ([ATP]i) by silica also decreased in a concentration-dependent manner. The relationship between [Ca2+]i and [ATP]i showed that [ATP]i depletion caused [Ca2+]i to rise. This study suggests that 1) the elevation of [Ca2+]i caused by silica is due mainly to influx through plasma membrane Ca2+ channel and non specific membrane damage (at high concentration) and 2) nephrotoxicity of silica shows site-specificity within the kidney.

Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice.

A transgenic mouse approach was used to examine the mechanism of principal cell-specific expression of aquaporin-2 (AQP2) within the renal collecting duct. RT-PCR and immunocytochemistry revealed that murine AQP2 was expressed in principal cells in the renal collecting duct, epithelial cells of the vas deferens, and seminiferous tubules within testis. The vas deferens expression was confirmed in rats. RT-PCR and immunocytochemistry showed that 14 kb of the human 5'-flanking region confers specific expression of a nucleus-targeted and epitope-tagged Cre recombinase in the principal cells within the renal collecting duct, in the epithelial cells of the vas deferens, and within the testis of transgenic mice. These results suggest that cell-specific expression of AQP2 is mediated at the transcriptional level and that 14 kb of the human AQP2 5'-flanking region contain cis elements that are sufficient for cell-specific expression of AQP2. Finally, renal principal cell expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of gene function in this cell type.

Intraepithelial Infiltration by Mast Cells with Both Connective Tissue-Type and Mucosal-Type Characteristics in Gut, Trachea, and Kidneys of IL-9 Transgenic Mice

IL-9 transgenic mice were analyzed for the presence of mast cells in different tissues. In these mice, increased mast cell infiltration was found in the gastric and intestinal epithelium as well as in the upper airways and kidney epithelium, but not in other organs, such as skin. IL-9 transgenic mast cells do not show signs of massive degranulation such as that found in IL-4 transgenic mice and are not involved in spontaneous pathologic changes. Gastric mast cells showed a phenotype related to connective-type mast cells, since they were stained by safranin, and strong expression of mouse mast cell protease-4 and -5 was found in this organ. However, they also expressed proteases related to the mucosal cell type, such as mouse mast cell protease-1 and -2. In vitro, although IL-9 by itself did not induce mast cell development from bone marrow progenitors, it strongly synergized with stem cell factor for the growth and differentiation of mast cells expressing the same protease pattern as that observed in IL-9 transgenic mice. Since constitutive stem cell factor expression was observed in vivo, and anti-c-Kit Abs inhibited IL-9 transgenic mastocytosis in the gut, this synergistic combination of factors is likely to be responsible for the mastocytosis observed in IL-9 transgenic mice. Taken together, these data demonstrate that IL-9 induces the in vivo amplification of a nonclassical mast cell subset with a mucosal localization but expressing proteases characteristic of both connective tissue-type and mucosal mast cells.

Apoptosis in kidneys of endothelin-1 transgenic mice.

Endothelin-1 (ET-1) transgenic mice are characterized by age-dependent development of renal cysts and renal fibrosis (interstitial fibrosis and glomerulosclerosis), leading to a progressive decrease in glomerular filtration rate. The mechanism causing the loss of functionally and morphologically normal renal tissue is unknown. An imbalance between cell proliferation and apoptosis in the kidneys of ET-1 transgenic mice might contribute to this process. We identified apoptotic cells in kidney sections by in situ end-labeling and by the typical nuclear chromatin morphology after propidium iodide (PI) staining. Cell proliferation was measured by estimating the proliferating cell nuclear antigen (PCNA)-positive cells. The numbers of apoptotic cells were significantly increased (p < 0.001) in the kidneys of 14-month-old ET-1 transgenic mice, whereas cell proliferation was not enhanced. Apoptotic cells were detected in the glomeruli, tubular cells, and renal interstitial cells in ET-1 transgenic mice. In conclusion, apoptotic loss of functional renal tissue appears to be associated with the progression of cyst formation and renal fibrosis. Therefore, an imbalance between cell proliferation and apoptosis could be an important cellular mechanism in ET-1 transgenic mice leading to end-stage kidney disease.

Electron Microscopic Localization of LacZ Expression in the Proximal Convoluted Tubular Cells of the Kidney in Transgenic Mice Carrying Chimeric Erythropoietin/lacZ Gene Constructs

Regulated expression of the erythropoietin (EPO) gene in the adult kidney plays a key role in the regulation of erythropoiesis. However, uncertainty exists regarding the type of kidney cells involved in EPO gene expression. We previously showed by light microscopy that the lacZ reporter gene is expressed and inducible by hypoxia/anemia in the proximal convoluted tubular (PCT) cells of the kidneys of transgenic mice carrying the 5′-lacZ construct, in which the lacZ gene was placed downstream of a 7.0-kb mouse EPO gene segment containing 6.5 kb of the 5′-flanking sequence. We, report here the light and transmission electron microscopic examination of lacZ expression in the kidneys of transgenic mice carrying the 5′-lacZ construct and two additional constructs carrying the 6.5-kb 5′-flanking sequence with the body of the gene alone, or along with the 1.2-kb 3′-flanking sequence. The electron microscopic analyses unequivocally demonstrated that lacZ under the regulatory control of the 6.5-kb 5′-flanking sequence with or without the body of the gene and the 1.2-kb 3′-flanking sequence was expressed predominantly in the proximal convoluted tubular cells of the kidney following hypoxia induction.

Establishment of human granulocyte‐macrophage colony stimulating factor producing transgenic SCID mice

Previous work has shown the usefulness of severe combined immunodeficient (SCID) mice as in vivo models for the growth of normal human haemopoietic cells and leukaemic cells. Many approaches have been made to improve the engraftment of human haemopoietic cells in SCID mice. We established transgenic mice producing human granulocyte-macrophage colony stimulating factor (hGM-CSF) with the homozygote of the scid gene. Endogenous serum hGM-CSF levels were detected by ELISA [mean 9585 pg/ml (line A, n = 4); mean 1610 pg/ml (line B, n = 4)]. Expression of hGM-CSF was observed in all organs tested including the heart, lung, liver, kidney, spleen, thymus, bone marrow and brain of hGM-CSF transgenic (hGMTg) mice. Morphological analysis of organs and peripheral blood cell counts showed no differences between hGMTg mice and their littermates. Murine Ba/F3 cells expressing functional hGM-CSF alpha beta receptor (BAF/alpha beta cells) could be successfully engrafted in hGMTg SCID mice. The cells invaded multiple organs and caused death within a few weeks of transplantation, although they infiltrated only the spleen of their littermates. These results showed that these hGM-CSF-producing SCID mice are useful as an in vivo assay system for investigating leukaemogenesis.

Stimulated secretion of lysosomal enzymes induced by drugs in transimmortalized proximal tubule mouse kidney cells.

We summarize the results of study of the properties of two models of transimmortalized proximal tubule epithelial cells derived from the kidneys of transgenic mice harboring the SV40 large T and little t antigens/L-pyruvate kinase hybrid gene. The two cell lines, referred to as PKSV-PCT and PKSV-PR cells, maintained for long-term passages the main biochemical and functional properties from the convoluted and terminal parts of the proximal tubule, respectively, from which they were derived. In PKSV-PCT cells, gentamicin induced lysosomal alkalinization, decreased the cellular N-acetyl-beta-D-glucuronidase, and stimulated its secretion in a dose-dependent manner. The results indicate that these models of mouse proximal cultured cells could be suitable models for the study of the cellular action of drugs.

Ten kilobases of 5′-flanking region confers proper regulation of the mouse alcohol dehydrogenase-1 ( Adh-1) gene in kidney and adrenal of transgenic mice

The expression profile of the mouse Adh-1 gene, which encodes class I alcohol dehydrogenase enzyme (ADH), is complex and includes tissue specificity and differential hormone responsiveness. Whereas kidney Adh-1 transcription rate is stimulated six- to sevenfold by testosterone treatment, adrenal gland ADH-1 mRNA is reduced to less than 5% of control level within 18 h following hormone administration. Androgen receptor is required for both responses since neither occurs in Tfm mutant mice lacking receptor. Hormonal and tissue-specific aspects of Adh-1 regulation were studied in transgenic mice harboring either of two constructs containing either −2.5 kb or −10 kb of 5′-flanking sequence attached to an Adh-1 minigene. The minigene transcript was expressed in kidney and adrenal tissues, but not liver, in five independent lines harboring a transgene with −2.5 kb of 5′-flanking sequence. Androgen treatment repressed the level of the minigene transcript in adrenal gland, but did not cause induction in kidney. In four lines of transgenic mice carrying the construct with −10 kb of 5′-flanking sequence, the minigene transcript was both repressed in adrenal and induced in kidney by testosterone. These lines have no detectable transgene expression in liver tissue. The −10 kb region in the mouse Adh-1 gene contains necessary controlling regions for proper tissue expression and hormonal regulation in kidney and adrenal; however, this region does not contain all essential elements necessary for expression in liver.

Differential expression of lacZ in the liver and kidney of transgenic mice carrying chimeric lacZ-erythropoietin gene constructs with or without its 1.2 kb 3'-flanking sequence.

Erythropoietin (EPO) plays a key role in erythropoiesis and is expressed predominantly in the fetal liver and in the adult kidney. The EPO gene is up-regulated at the transcriptional level under hypoxic/anemic conditions. We studied the role of the 5'- and 3'-flanking sequences of the mouse EPO gene in its tissue-specific and hypoxia-induced expression by developing transgenic mouse lines carrying chimeric EPO-lacZ gene constructs. Transgenic mice carrying a 6.5 kb segment of the 5'-sequence and most of the EPO gene in which lacZ was substituted for exon 1 (5'-lacZ-EPO) demonstrated induction of lacZ expression following hypoxia/ anemia induction in both the liver and kidney of adult mice. However, transgenic mice carrying the above construct along with the 1.2 kb 3'-flanking sequence (5'-lacZ-EPO-3') showed a high level of lacZ expression following hypoxia/anemia induction in adult kidney but not in adult liver. With the aim of further understanding the role of the 3'-flanking sequence in tissue-specific expression of the EPO gene, we studied the interactions of protein factors with this 1.2 kb 3' region and demonstrated that multiple sets of protein factors interact tissue specifically with a 10 bp sequence, TCAAAGATGG, located downstream of the previously characterized 3' hypoxia-responsive enhancer element.

Renal nitric oxide synthases in transgenic sickle cell mice

The alpha H beta S [beta MDD] mouse is a useful model for studying renal functional abnormalities in sickle cell disease. We previously reported that these mice develop a urine concentrating defect when chronically exposed to a low oxygen environment. In the present study, we measured glomerular filtration rate (GFR), urinary excretion of NO2 s+ NO3, the stable products of nitric oxide (NO), and the abundance of endothelial constitutive nitric oxide synthase (NOS III) and inducible nitric oxide synthase (NOS II) in the kidneys by Western blot. Immunohistochemistry was also carried out. We found that GFR is significantly higher in the transgenic mice than in controls. The urinary NO2 + NO3/creatinine ratio was also higher. The Western blots revealed that both NOS III and NOS II are markedly increased in the kidneys of transgenic mice as compared to normal control mice. Immunohistochemistry localized NOS III reactivity in proximal convoluted cells in the cortex of control and alpha H beta S [beta MDD] mice. NOS II immunostaining was not seen in control mice but was clearly evident in glomeruli and distal nephron segments of the alpha H beta S [beta MDD] mice. These observations suggest that NOS II is induced in glomeruli and distal nephrons of the alpha H beta S [beta MDD] mice. An increase in synthesis of NO may occur in the glomeruli as a result of NOS II induction, and this may contribute to the hyperfiltration in these mice.

CHARACTERIZATION OF HUMAN RENIN TRANSGENE RESPONSE TO INCREASED CIRCULATING ANGIOTENSIN II AND HYPERTENSION IN TRANSGENIC MICE. • 2209

We have described a paradoxical upregulation of human renin mRNA in the kidney of transgenic mice containing both human renin and angiotensinogen genes (double transgenic). These mice exhibit hypertension and increased levels of angiotensin II (AII). In contrast, expression of the endogenous mouse renin gene decreased as expected in response to these stimuli.

Relationships between intermediate filaments and cell-specific functions in renal cell lines derived from transgenic mice harboring the temperature-sensitive T antigen

Four renal cell lines were derived from glomeruli, proximal, distal, and cortical collecting tubules microdissected from the kidneys of transgenic mice carrying the temperature-sensitive mutant of the simian virus 40 large T antigen under the control of the vimentin promoter. All four cell lines contained large T antigen in their nuclei, grew rapidly, and contained vimentin filaments when grown in serum-enriched medium at the permissive temperature of 33 degrees C. The glomerular cell line formed multiple layers of cells and contained smooth muscle actin and desmin filaments, features of mesangial cells. The three tubule cell lines formed monolayers of polarized cuboid cells separated by tight junctions and having a patchy distribution of cytokeratins K8-K18. A shift from 33 degrees C to the restrictive temperature (39.5 degrees C) stopped cell growth in all cell lines and caused profound changes in the content of intermediate filaments. Vimentin was still present in mesangial-like cells, but the proximal, distal, and collecting tubule cells contained uniform networks of cytokeratins K8-K18 and desmoplakin I and II around the cell peripheries. Potassium transport, mediated by Na+-K+ ATPase pumps and specific cAMP hormonal sensitivities, significantly increased in proximal, distal, and collecting tubule cells when shifted from 33 degrees C to 39.5 degrees C. Thus, the temperature-dependent inactivation of large T antigen, responsible for the arrest of cell growth, did not affect the phenotype of mesangial-like glomerular cells but induced some changes in the expression of intermediate filaments and restored, at least partially, the main parental cell-specific functions in proximal, distal, and collecting tubule cultured cells.

Protection from complement‐mediated injury in livers and kidneys of transgenic mice expressing human complement regulators

Abstract: The major problem in the use of phylogenetically distant donors is a fast, strong reaction called hyperacute rejection. This reaction mediated by complement is directed against the vascular endothelia of the transplanted organ. Complement activation is tightly controlled by several regulatory proteins which inhibit the formation and function of different complement components. To verify the hypothesis that organs expressing such inhibitory factors could be spared from complement‐mediated hyperacute rejection, we have generated mice transgenic for the human complement inhibitor membrane cofactor protein (hMCP) and decay accelerating factor (hDAF). Different levels of hMCP and/or hDAF expression, according to the promoter used, were detected by RNA analysis in the major organs, specifically on the organ vascular endothelia, as revealed by immunohistochemical analysis. The development of an in vivo model of human plasma perfusion allowed the characterization of complement‐mediated damage in control animals and the degree of protection due to the presence of hMCP, hDAF, or both in the organs derived from single or double transgenic mice. In this paper we compare the level of expression of complement regulators with the degree of protection in two major organs: liver and kidney.

Aflatoxin B1 induced lacI mutation in liver and kidney of transgenic mice C57BL/6N: effect of phorone.

Transgenic C57BL/6N mice containing a lambda shuttle vector carrying a lacI target and an alpha-lacZ reporter gene have been used to study the modulating effect of phorone, a glutathione-depleting agent, on the mutagenic activity of aflatoxin B1 (AFB1) in vivo. Animals were treated with AFB1 (8 mg/kg) for four consecutive days and the animals sacrificed 21 days after the last treatment. Treatment with AFB1 alone did not result in a significant increase in mutation frequency in the liver and kidney. When the animals were treated with phorone 4 h prior to treatment with AFB1 a significant increase in mutation frequency was observed in the liver (4-fold) and kidney (1.5-fold). Phorone treatment did not increase the AFB1-induced mutation frequency in the lung and intestine. DNA sequence analyses of 30 independent clones isolated from the liver of AFB1-treated animals showed that G:C --> T:A transversion (60%) was the predominant mutational event. Mutations within the lacI gene could not be detected in seven of 30 mutants. The mutations were randomly distributed throughout the coding sequences of the lacI gene and no hotspots for the mutations were observed. However, codons 86 and 928 appeared to be major sites for mutation. The study shows that the transgenic mouse in vivo mutagenesis model can be used to study the influence of effect-modifying compounds on the mutagenic activity of known carcinogens.

A 700-bp fragment of the human antithrombin III promoter is sufficient to confer high, tissue-specific expression on human apolipoprotein A-II in transgenic mice

The human antithrombin III-encoding gene (h AT-III) promoter ( ph AT-III) was used to generate transgenic mice producing a human hepatic apolipoprotein, apolipoprotein A-II (hApoA-II). Integration of the transgene into the mouse genome resulted in the efficient production of hApoA-II in plasma, reaching up to 0.40 g/l in two transgenic lines. The human ApoA-II mRNA was detected at high levels, both in the liver and in the kidney of transgenic mice. The rat AT-III gene shows the same expression pattern. In contrast, as previously described, the same promoter permitted the expression of the SV40 large T antigen only in the liver of transgenic mice. In view of the extra-hepatic distribution of the ApoA-II mRNA, a preliminary characterization of the hAT-III proximal promoter ( phAT-III), driving the expression of the transgene, was realized. Using DNase I footprinting analysis with liver nuclear extracts, four protected regions (I–IV) were identified in the first 175 bp of the 5′ region of h AT-III. Electrophoretic mobility shift assays performed with liver and kidney nuclear extracts indicate that region III (nucleotides (nt) −67 to −90) interacts with the liverenriched HNF4 nuclear factor. Furthermore, our data suggest that region I (nt −123 to −138) interacts with the liverenriched HNF3 transcription factor family, both in liver and kidney. Taken together, these results demonstrate that ph AT-III is a useful tool to create transgenic mice producing high plasma levels of a human apolipoprotein due to expression of the transgene in liver and kidney. Interestingly, our data suggest that, in both organs, common pathways are involved in the control of ph AT-III activity

Cortical expression of the human angiotensinogen gene in the kidney of transgenic mice

We have previously generated "Tsukuba hypertensive mice" with elevated blood pressure by cross-mating separate lines of transgenic animals carrying either 15 kb of the human renin gene including its native 3-kb promoter or 14 kb of the human angiotensinogen gene along with its 1.3-kb promoter, the former of which is expressed predominantly in the kidney and the latter of which is also expressed in the kidney to levels comparable to those found in the liver. To investigate whether the integrated human angiotensinogen gene is prominently expressed in the kidney of transgenic mice, we have analyzed a production region of the transgene mRNA by in situ hybridization technique. This analysis clearly demonstrated that human angiotensinogen mRNA is localized specifically to the cortex region of transgenic mouse kidney. The present finding indicates a possible involvement of the renal renin-angiotensin system in the pathogenesis of high blood pressure in transgenic mice.

Functional expression of the human angiotensinogen gene in transgenic mice.

The renin-angiotensin system is a major determinant of arterial pressure and volume homeostasis in mammals through the actions of angiotensin II, the proteolytic digestion product of angiotensinogen. Molecular genetic studies in several human populations have revealed genetic linkage between the angiotensinogen gene and both hypertension and increased plasma angiotensinogen. Transgenic mice were generated with a human angiotensinogen genomic clone to develop an animal model to examine tissue- and cell-specific expression of the gene and to determine if overexpression of angiotensinogen results in hypertension. Human angiotensinogen mRNA was expressed in transgenic mouse liver, kidney, heart, adrenal gland, ovary, brain, and white and brown adipose tissue and, in kidney, was exclusively localized to epithelial cells of the proximal convoluted tubules. Plasma levels of human angiotensinogen were approximately 150-fold higher in transgenic mice than that found normally in human plasma. The blood pressure of mice bearing the human angiotensinogen gene was normal but infusion of a single bolus dose of purified human renin resulted in a transient increase in blood pressure of approximately 30 mm Hg within 2 min. These results suggest that abnormalities in the angiotensinogen gene resulting in increased circulating levels of angiotensinogen could potentially contribute in part to the pathogenesis of essential hypertension.

D-Limonene Induced Hyaline Droplet Nephropathy in α2u-Globulin Transgenic Mice

d-Limonene Induced Hyaline Droplet Nephropathy in α2u-Globulin Transgenic Mice. Lehmian-McKeeman, L. D., and Caudill, D. (1994). Fundam. Appl. Toxicol. 23, 562-568. d-Limonene is a hyaline droplet inducing agent and produces nephrotoxicity in male rats when the 1,2 epoxide metabolite binds to α2u-globulin. Mice, which do not synthesize α2u-globulin, are resistant to hyaline droplet nephropathy. In this study, the ability of d -limonene to cause hyaline droplet nephropathy in a transgenic mouse engineered to express α2u-globulin was evaluated. The C57BL/6 derived mice excreted 0.4 ± 0.1 mg α2u-globulin/day, or approximately 16 mg α2u-globulin/kg body wt. This represents about 30% or the amount excreted by adult male rats (11.9 ± 1.1 mg/day or approximately 48 mg/kg). Transgenic mice excreted less mouse urinary protein (9.3 ± 1.2 mg/day) than normal mice (15.1± 1.6 mg/day). Unlike normal male rats, untreated transgenic mice did not show significant spontaneous hyaline droplet formation. Liver microsomes from naive transgenic mice oxidized d -limonene to the cis- and trans-isomers of the 1,2-epoxide, and following oral treatment with [ 14C] d-limonene reversible binding of d-limonene equivalents to renal cytosolic proteins was observed. Furthermore, with d -limonene treatment, hyaline droplets were observed in the transgenic mouse kidneys. These droplets, however, were much smaller in size than those seen in d-limonene-treated male rats. The accumulation of α2u-globulin in the kidneys of transgenic mice and normal male rats before and after d-limonene treatment was analyzed by Western blotting. These results indicated that α2u-globulin was present in the kidneys of the control transgenic mice, despite the lack of spontaneous hyaline droplet formation. After d-limonene treatment, approximately a threefold increase in α2u-globulin in the transgenic mouse kidney was observed, a response similar in magnitude to that seen in d-limonene-treated male rats. These results indicate that expression of α2u-globulin in a species that does not normally develop hyaline droplet nephropathy is necessary and sufficient to render that species sensitive to this renal toxicity.

An analysis of regulatory elements in the phosphoenolpyruvate carboxykinase (GTP) gene which are responsible for its tissue-specific expression and metabolic control in transgenic mice.

Sequences in the gene for P-enolpyruvate carboxykinase (PEPCK) which are responsible for its complex pattern of transcriptional control were determined using transgenic mice containing a chimeric PEPCK-bovine growth hormone (bGH) gene consisting of a segment of the PEPCK promoter from -460 to +73, with mutations in specific regulatory domains. A mutation in the cAMP response element (CRE) (-87 to -74), which binds CCATT/enhancer-binding protein beta (C/EBP beta) and/or cAMP response element-binding protein (CREB), resulted in a 4- and 20-fold elevation in the level of bGH mRNA in the liver and kidney of transgenic mice, respectively. Expression of the PEPCK-bGH gene in the liver was reduced 60% by a mutation in the P3 (I) region (-248 to -230), whereas expression in the kidney was increased 10-fold by the same mutation. A mutation in the P2 region (-200 to -164) greatly reduced expression of the PEPCK-bGH gene in the kidney but not in the liver. Induction of hepatic PEPCK-bGH gene expression by Bt2cAMP was eliminated by mutations in the CRE, P1, P3(I), or by a double mutation of the CRE and P3(I). Mutations in the CRE or P3(I) regions of the PEPCK promoter did not interfere with the expected induction of the PEPCK-bGH gene in the liver at birth. None of the mutations in the PEPCK promoter interfered with the induction of transcription of the PEPCK-bGH gene in the liver when mice were fed a carbohydrate-free diet or the deinduction of transcription from the PEPCK promoter caused by a diet high in carbohydrate, whereas a mutation in P2 (an HNF-1 binding domain) eliminated dietary regulation of transcription of the transgene in the kidney. A model to explain the role of the various elements in the PEPCK promoter on the control of PEPCK gene transcription in the liver and kidney is presented.

Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis

Two cell lines, TgSVA and TgSVB, were established from the kidneys of transgenic mice carrying the human gene encoding poliovirus receptor. The cells were highly susceptible to poliovirus infection, and a large amount of infectious particles was produced in the infected cells at 37 degrees C. However, the virus yield was greatly reduced at 40 degrees C. This phenomenon was common to all mouse cells tested. To identify the temperature-sensitive step(s) of the virus infection cycle, different steps of the infection cycle were examined for temperature sensitivity. The results strongly suggested that the growth restriction observed at 40 degrees C was due to reduced efficiency of the initiation process of virus-specific RNA synthesis. Furthermore, this restriction appeared to occur only on the synthesis of positive-strand RNA. Virus-specific RNA synthesis in crude replication complexes was not affected by the nonpermissive temperature of 40 degrees C. In vitro uridylylation of VPg seemed to be temperature sensitive only after prolonged incubation at 40 degrees C. These results indicate that a specific host factor(s) is involved in the efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperature sensitive in TgSVA and TgSVB cells.