Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels.

A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines.

Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼ 80% of mutants showed specific staining in one or more tissues, while ∼ 20% showed no specific staining, ∼ 13% had staining in only one tissue, and ∼ 25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼ 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known.

Applying the ARRIVE Guidelines to an In Vivo Database.

The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were developed to address the lack of reproducibility in biomedical animal studies and improve the communication of research findings. While intended to guide the preparation of peer-reviewed manuscripts, the principles of transparent reporting are also fundamental for in vivo databases. Here, we describe the benefits and challenges of applying the guidelines for the International Mouse Phenotyping Consortium (IMPC), whose goal is to produce and phenotype 20,000 knockout mouse strains in a reproducible manner across ten research centres. In addition to ensuring the transparency and reproducibility of the IMPC, the solutions to the challenges of applying the ARRIVE guidelines in the context of IMPC will provide a resource to help guide similar initiatives in the future.

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein.

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9-FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.

Positional cloning of the combined hyperlipidemia gene Hyplip1.

Familial combined hyperlipidemia (FCHL, MIM-144250) is a common, multifactorial and heterogeneous dyslipidemia predisposing to premature coronary artery disease and characterized by elevated plasma triglycerides, cholesterol, or both. We identified a mutant mouse strain, HcB-19/Dem (HcB-19), that shares features with FCHL, including hypertriglyceridemia, hypercholesterolemia, elevated plasma apolipoprotein B and increased secretion of triglyceride-rich lipoproteins. The hyperlipidemia results from spontaneous mutation at a locus, Hyplip1, on distal mouse chromosome 3 in a region syntenic with a 1q21-q23 FCHL locus identified in Finnish, German, Chinese and US families. We fine-mapped Hyplip1 to roughly 160 kb, constructed a BAC contig and sequenced overlapping BACs to identify 13 candidate genes. We found substantially decreased mRNA expression for thioredoxin interacting protein (Txnip). Sequencing of the critical region revealed a Txnip nonsense mutation in HcB-19 that is absent in its normolipidemic parental strains. Txnip encodes a cytoplasmic protein that binds and inhibits thioredoxin, a major regulator of cellular redox state. The mutant mice have decreased CO2 production but increased ketone body synthesis, suggesting that altered redox status down-regulates the citric-acid cycle, sparing fatty acids for triglyceride and ketone body production. These results reveal a new pathway of potential clinical significance that contributes to plasma lipid metabolism.

Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in immunity, inflammation, angiogenesis, and cancer. S1P lyase (SPL) is the essential enzyme responsible for S1P degradation. SPL augments apoptosis and is down-regulated in cancer. SPL generates a S1P chemical gradient that promotes lymphocyte trafficking and as such is being targeted to treat autoimmune diseases. Despite growing interest in SPL as a disease marker, antioncogene, and pharmacological target, no comprehensive characterization of SPL expression in mammalian tissues has been reported. We investigated SPL expression in developing and adult mouse tissues by generating and characterizing a ß-galactosidase-SPL reporter mouse combined with immunohistochemistry, immunoblotting, and enzyme assays. SPL was expressed in thymic and splenic stromal cells, splenocytes, Peyer's Patches, colonic lymphoid aggregates, circulating T and B lymphocytes, granulocytes, and monocytes, with lowest expression in thymocytes. SPL was highly expressed within the CNS, including arachnoid lining cells, spinal cord, choroid plexus, trigeminal nerve ganglion, and specific neurons of the olfactory bulb, cerebral cortex, midbrain, hindbrain, and cerebellum. Expression was detected in brown adipose tissue, female gonads, adrenal cortex, bladder epithelium, Harderian and preputial glands, and hair follicles. This unique expression pattern suggests SPL has many undiscovered physiological functions apart from its role in immunity.

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction.

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

In vivo imaging of tissue eosinophilia and eosinopoietic responses to schistosome worms and eggs.

Using a sensitive transgenic reporter mouse system and in vivo biophotonic imaging techniques, we present a dynamic analysis of eosinophil responses to schistosome infection. Use of this methodology provided previously unattainable detail on the spatial and temporal distribution of tissue eosinophilia and eosinopoietic responses to schistosome worms and eggs. Dramatic hepatic and intestinal eosinophilia in response to the deposition of schistosome eggs, with accompanying eosinopoiesis in the bone marrow, was observed between weeks 8 and 10 p.i., with subsequent downregulation evident by week 11. Contrary to expectations, we also demonstrate that schistosome parasites themselves induce significant intestinal eosinophilia and eosinopoiesis in the bone marrow at very early stages during prepatent infection.

Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.

Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.

A Cyp1a2-luciferase transgenic CD-1 mouse model: responses to aryl hydrocarbons similar to the humanized AhR mice.

Here we describe a transgenic mouse model [Crl:CD-1(ICR)BR-Tg(Cyp1a2-luc)Xen] using luciferase as a reporter for Cyp1a2 gene regulation. An 8.4-kilobase mouse Cyp1a2 promoter driving the firefly luciferase gene was microinjected into single-cell-stage CD-1 mouse embryos. A transgenic mouse line was selected based on basal and induced levels of the transgene in mouse liver by an in vivo bioluminescent imaging method. The basal levels of the luciferase reporter in liver were expressed much higher than other tissues, which correlated well with the endogenous Cyp1a2 mRNA tissue distribution. Male signals were about 23-fold higher than females in liver. However, the Cyp1a2 mRNA showed no gender difference. When mice were challenged with xenobiotics, the liver luciferase signal was induced to various degrees. At the doses we used, the relative effects were phenobarbital > 2,3,7,8-tetrachlorodibenzo-p-dioxin > 3-methylcholanthrene > benzo[a]pyrene and beta-naphthoflavone. Induction of the Cyp1a2-luc reporter was generally consistent with the endogenous Cyp1a2 mRNA. However, phenobarbital induction was unexpectedly higher, while beta-naphthoflavone induction of the reporter was much lower than that of the endogenous Cyp1a2 gene. Induction of the Cyp1a2-luc transgene by aryl hydrocarbons (Ah) in the CD-1 background was much less than that found in the Ah responsive C57BL/6 mice, while being similar to the nonresponsive DBA/2 strain. Sequence analysis of the CD-1 Ah receptor (AhR) cDNA clones demonstrated that consensus sequence was identical to some of the Ah-responsive strains such as BALB/C and CBA/J mice. The 104-kD AhR protein was not detectable in CD-1 mice, while the 97-kD AhR was detected in the C57BL/6 mice by Western blot using an AhR antibody. Low expression of the AhR in CD-1 mice could be in part responsible for low responsiveness to Ah compounds. The findings demonstrated the outbred CD-1 mouse is a low-responsive strain, and the Cyp1a2-luc transgenic CD-1 mice can be used for studying the regulation of the mouse Cyp1a2 gene in an Ah low-responsive strain in real time using the bioluminescent imaging approach.

In vivo monitoring of pancreatic beta-cells in a transgenic mouse model.

We generated a transgenic mouse model (RIP-luc) for the in vivo monitoring of pancreatic islet mass and function in response to metabolic disease. Using the rat insulin promoter fused to firefly luciferase, and noninvasive technology to detect luciferase activity, we tracked changes in reporter signal during metabolic disease states and correlated the changes in luciferase signal with metabolic status of the mouse. Transgene expression was found to be specific to the pancreatic islets in this transgenic model. Basal transgene expression was tracked in male and female mice fed either a chow or a high-fat diet and in response to treatment with streptozotocin. Pancreatic bioluminescent signal increased in mice fed a high-fat diet compared with chow-fed animals. In a model of chemically induced diabetes, the bioluminescent signal decreased in accordance with the onset of diabetes and reduction of islet beta-cell number. Preliminary studies using islets transplanted from this transgenic model suggest that in vivo image analysis can also be used to monitor transplanted islet viability and survival in the host. This transgenic model is a useful tool for in vivo studies of pancreatic beta-cells and as a donor for islet transplantation studies.

Visualizing drug efficacy in vivo.

Many enzymes are therapeutic targets for drug discovery, whereas other enzymes are important for understanding drug metabolism and pharmacokinetics during compound testing in animals. Testing of drug efficacy and metabolism in an animal model requires the measurement of disease endpoints as well as assays of enzyme activity in specific tissues at selected time points during treatment. This requires the removal of tissue and biochemical assays. Techniques to noninvasively assess drug effects on enzyme activity using imaging technology would facilitate understanding of drug efficacy, pharmacokinetics, and drug metabolism. Using a commercially available cytochrome P-450 3A substrate whose oxidized product is a luciferase substrate, we show for the first time that cytochrome P-450 enzyme activity can be measured in vivo in real time by bioluminescent imaging. This imaging approach could be applicable to study drug effects on therapeutic target enzymes, as well as drug metabolism enzymes.

Serum amyloid A-luciferase transgenic mice: response to sepsis, acute arthritis, and contact hypersensitivity and the effects of proteasome inhibition.

Acute phase serum amyloid A proteins (A-SAAs) are multifunctional apolipoproteins produced in large amounts during the acute phase of an inflammation and also during the development of chronic inflammatory diseases. In this study we present a Saa1-luc transgenic mouse model in which SAA1 gene expression can be monitored by measuring luciferase activity using a noninvasive imaging system. When challenged with LPS, TNF-alpha, or IL-1beta, in vivo imaging of Saa1-luc mice showed a 1000- to 3000-fold induction of luciferase activity in the hepatic region that peaked 4-7 h after treatment. The induction of liver luciferase expression was consistent with an increase in SAA1 mRNA in the liver and a dramatic elevation of the serum SAA1 concentration. Ex vivo analyses revealed luciferase induction in many tissues, ranging from several-fold (brain) to >5000-fold (liver) after LPS or TNF-alpha treatment. Pretreatment of mice with the proteasome inhibitor bortezomib significantly suppressed LPS-induced SAA1 expression. These results suggested that proteasome inhibition, perhaps through the NF-kappaB signaling pathway, may regulate SAA1 expression. During the development of acute arthritis triggered by intra-articular administration of zymosan, SAA1 expression was induced both locally at the knee joint and systemically in the liver, and the induction was significantly suppressed by bortezomib. Induction of SAA1 expression was also demonstrated during contact hypersensitivity induced by topical application of oxazolone. These results suggest that both local and systemic induction of A-SAA occur during inflammation and may contribute to the pathogenesis of chronic inflammatory diseases associated with amyloid deposition.

NF-kappaB and not the MAPK signaling pathway regulates GADD45beta expression during acute inflammation.

The GADD45 (growth arrest and DNA damage-inducible) family of genes is involved in the regulation of cell cycle progression and apoptosis. To study signaling pathways affecting GADD45beta expression and to examine systematically in vivo the GADD45beta expression in tissues following various toxic stresses, we created a transgenic mouse by fusing the GADD45beta promoter to firefly luciferase (Gadd45beta-luc). In vivo GADD45beta expression was assessed by measuring the luciferase activity in the Gadd45beta-luc transgenic mouse using a non-invasive imaging system (IVIS Imaging System, Xenogen Corporation). We found that a number of agents that induce oxidative stress, such as sodium arsenite, CCl4, lipopolysaccharide (LPS), or tumor necrosis factor-alpha, are able to induce luciferase expression throughout the entire animal. In liver, spleen, lung, intestine, kidney, and heart, we observed an induction of luciferase activity after LPS treatment, which correlates with an increase of GADD45beta mRNA in these tissues. Processes that induce DNA damage activate the NF-kappaB signaling pathway. Several inhibitors of the NF-kappaB signaling pathway, including dexamethasone, thalidomide, and a proteasome inhibitor, bortezomib, showed inhibitory effects on LPS-induced GADD45beta expression as indicated by a decrease of the luciferase activity. Northern blot analysis confirmed a broad inhibitory effect of bortezomib on LPS-induced GADD45beta mRNA expression in spleen, lung, and intestine. In liver of bortezomib-treated mice, we observed a reverse correlation between the luciferase activity and the GADD45beta mRNA level. We speculate that such a discrepancy could be due to severe liver toxicity caused by bortezomib and LPS co-treatment. MAPK inhibitors had transient and inconsistent effects on LPS-induced luciferase expression. Our data are consistent with the notion that NF-kappaB, but not the MAPK signaling pathways, is involved in the in vivo regulation of GADD45beta expression. Thus, NF-kappaB signaling involves induction of GADD45beta expression, which supports the proposed role of GADD45beta in protecting cells against DNA damaged under various stress conditions.

Regulation of IkappaBalpha expression involves both NF-kappaB and the MAP kinase signaling pathways.

IkappaBalpha is an inhibitor of the nuclear transcription factor NF-kappaB. Binding of IkappaBalpha to NF-kappaB inactivates the transcriptional activity of NF-kappaB. Expression of IkappaBalpha itself is regulated by NF-kappaB, which provides auto-regulation of this signaling pathway. Here we present a mouse model for monitoring in vivo IkappaBalpha expression by imaging IkappaBalpha-luc transgenic mice for IkappaBalpha promoter driven luciferase activity. We demonstrated a rapid and systemic induction of IkappaBalpha expression in the transgenic mice following treatment with LPS. The induction was high in liver, spleen, lung and intestine and lower in the kidney, heart and brain. The luciferase induction in the liver correlated with increased IkappaBalpha mRNA level. Pre-treatment with proteasome inhibitor bortezomib dramatically suppressed LPS-induced luciferase activity. The p38 kinase inhibitor SB203580 also showed moderate inhibition of LPS-induced luciferase activity. Analysis of IkappaBalpha mRNA in the liver tissue showed a surprising increase of the IkappaBalpha mRNA after bortezomib and SB203580 treatments, which could be due to increased IkappaBalpha mRNA stability. Our data demonstrate that regulation of IkappaBalpha expression involves both the NF-kappaB and the p38 signaling pathways. The IkappaBalpha-luc transgenic mice are useful for analyzing IkappaBalpha expression and the NF-kappaB transcriptional activity in vivo.

Differential regulation of the human CYP3A4 promoter in transgenic mice and rats.

Previously we described a transgenic mouse model [FVB/NTg(CYP3A4-luc)Xen] using a reporter construct consisting of 13 kilobases of the human CYP3A4 promoter driving the firefly luciferase gene in the inbred FVB/N mouse strain. Here we report regulation of the same CYP3A4-luc reporter gene in a transgenic outbred mouse strain (CD-1) and in a transgenic rat (Sprague-Dawley). Basal reporter expression and responses to several xenobiotics in the transgenic CD-1 mice [CD-1/Crl-Tg(CYP3A4-luc)Xen] were similar to those in the transgenic FVB/N mice. In both mouse backgrounds, the basal levels of the reporter were higher in male compared with female, and in the FVB/N strain there was greater induction for all drugs in male compared with female; however, in the CD-1 background this gender difference for induction was not obvious. In contrast with transgenic mice, transgenic rats [SD/Tac-Tg(CYP3A4-luc)Xen] expressed the luciferase reporter at higher basal levels in female compared with male rats. Responses to some compounds were much greater in rats than in mice, and the kinetics of induction was different with peak induction occurring later in the rat compared with the mouse. Our results suggest that the human CYP3A4 promoter is regulated differently in transgenic mice and rats in some aspects.

Tracking angiogenesis induced by skin wounding and contact hypersensitivity using a Vegfr2-luciferase transgenic mouse.

The vascular endothelial growth factor-2 (VEGFR2) gene is transcriptionally regulated during angiogenesis. The ability to monitor and quantify VEGFR2 expression in vivo may facilitate a better understanding of the role of VEGFR2 in different states. Here we describe a transgenic mouse, Vegfr2-luc, in which a luciferase reporter is under control of the murine VEGFR2 promoter. In adult mice, luciferase activity was highest in lung and uterus, intermediate in heart, skin, and kidney, and lower in other tissues. Luciferase expression in these tissues correlated with endogenous VEGFR2 mRNA expression. In a cutaneous wound-healing model, Vegfr2-luc expression was induced in the wound tissue. Histologic and immunohistochemical studies showed significant macrophage infiltration into the wound and induction of Vegfr2-luc expression in endothelial and stromal cells. Dexamethasone significantly suppressed Vegfr2-luc expression and macrophage infiltration into the wound, resulting in delayed healing and impaired angiogenesis. In a skin hypersensitivity reaction produced by treatment with oxazolone, Vegfr2-luc expression was induced in the ear. Treatment by dexamethasone markedly suppressed Vegfr2-luc expression and leukocyte infiltration in the ear and was correlated with reduced dermal edema and epidermal hyperplasia. The Vegfr2-luc model will be valuable in monitoring the ability of drugs to affect angiogenesis in vivo.

An inducible nitric oxide synthase-luciferase reporter system for in vivo testing of anti-inflammatory compounds in transgenic mice.

The inducible NO synthase gene (iNOS) plays a role in a number of chronic and acute conditions, including septic shock and contact hypersensitivity autoimmune diseases, such as rheumatoid arthritis, gastrointestinal disorders, and myocardial ischemia. The iNOS gene is primarily under transcriptional control and is induced in a variety of conditions. The ability to monitor and quantify iNOS expression in vivo may facilitate a better understanding of the role of iNOS in different diseases. In this study, we describe a transgenic mouse (iNos-luc) in which the luciferase reporter is under control of the murine iNOS promoter. In an acute sepsis model produced by injection of IFN-gamma and LPS, we observed an induction of iNOS-driven luciferase activity in the mouse liver. This transgene induction is dose and time dependent and correlated with an increase of liver iNOS protein and iNOS mRNA levels. With this model, we tested 11 compounds previously shown to inhibit iNOS induction in vitro or in vivo. Administration of dexamethasone, epigallocatechin gallate, alpha-phenyl-N-tert-butyl nitrone, and ebselen significantly suppressed iNOS transgene induction by IFN-gamma and LPS. We further evaluated the use of the iNos-luc transgenic mice in a zymosan-induced arthritis model. Intra-articular injection of zymosan induced iNos-luc expression in the knee joint. The establishment of the iNos-luc transgenic model provides a valuable tool for studying processes in which the iNOS gene is induced and for screening anti-inflammatory compounds in vivo.

Diet-induced obesity and hepatic gene expression alterations in C57BL/6J and ICAM-1-deficient mice.

The effects of high-fat feeding on the development of obesity were evaluated in intercellular adhesion molecule-1 (ICAM-1) knockout and C57BL/6J (B6) male mice fed a high-fat diet for < or =50 days. Serum and tissues were collected at baseline and after 1, 11, and 50 days on the diet. After 11 days on the diet, ICAM-1-deficient, but not B6, mice developed fatty livers and showed a significant increase in inguinal fat pad weight. At day 50, ICAM-1-deficient mice weighed less, and their adiposity index and circulating leptin levels were significantly lower than those of B6 controls. To better understand the early differential response to the diet, liver gene expression was analyzed at three time points by use of Affymetrix GeneChips. In both strains, a similar pattern of gene expression was detected in response to the high-fat diet. However, sterol regulatory element-binding protein-1, apolipoprotein A4, and adipsin mRNAs were significantly induced in ICAM-1-deficient livers, suggesting that these genes and their associated pathways may be involved in the acute diet response observed in the knockout mice.