SEARCH · PubMed Health

Results for “Reporter genes”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

PubMed2002

The first reporter gene assay on living cells: green fluorescent protein as reporter gene for the investigation of Gi-protein coupled receptors.

Reporter gene assay systems are important tools for the investigation of G-protein coupled receptors and their interaction with ligands. Here, we describe a novel reporter gene assay system for the investigation of Gi-protein coupled receptors in living cells. For the first time green fluorescent protein (GFP) was used as reporter gene under the transcriptional control of cAMP-response elements (CREs). Three different reporter gene vectors with increasing numbers of CREs were cloned and GFP expression was investigated after forskolin stimulation. Furthermore, the novel reporter system was successfully applied to the neuropeptide Y (NPY) rY5 receptor subtype, a Gi-protein coupled receptor. Our data clearly demonstrate dose-dependent GFP expression for NPY. Furthermore, receptor subtype selectivity of well characterized NPY analog could be proven for the NPY rY5 receptor. The great advantage of the method is that no cell lysis is required and assays can be performed on living cells. Accordingly, in vitro testing of agonist gets faster and significantly more convenient.

Animals↗

PubMed2005

Noninvasive monitoring of target gene expression by imaging reporter gene expression in living animals using improved bicistronic vectors.

UNLABELLED: Indirect, noninvasive imaging of therapeutic gene expression based on levels of reporter gene expression is a powerful tool to devise improved therapeutic strategies in cancer gene therapy. The use of bicistronic vectors carrying internal ribosome entry sites (IRESs) allows the coexpression of multiple gene products from the same promoter but leads to considerable attenuation of the downstream gene. In this study, we describe the use of 10 linked copies of the Gtx (homeodomain protein) IRES (abbreviated as SIRES) in place of the encephalomyocarditis (EMCV) IRES in mediating downstream reporter gene expression in cell culture and in vivo. METHODS: We constructed several plasmid vectors carrying different upstream and downstream reporter genes (herpes simplex virus type I thymidine kinase [tk], firefly luciferase [fl], and Renilla luciferase [rl]) placed between EMCV IRES and SIRES segments. RL, FL, and TK enzyme activities in N2a, C6, and 293 cells transiently transfected with these vectors were found to be significantly higher for the SIRES vectors than for the EMCV IRES vectors. For in vivo experiments, 4 stably transfected N2a cell lines were implanted in nude mice. The mice were imaged for rl and fl gene expression using a charged-coupled device (CCD) camera. For bioluminescence and microPET imaging of downstream gene expression of fl and tk genes, respectively, mice carrying 4 stably transfected xenografts were imaged using the CCD camera and microPET. RESULTS: In cell culture, using rl as the upstream gene, we demonstrate that the expression of the downstream tk gene is 12-fold greater using SIRES when compared with EMCV IRES. Furthermore, the expression of the 2 genes was highly correlated in N2a cells. In vivo bioluminescence imaging using 4 stably transfected N2a cell lines revealed increasing levels of rl and fl gene expression. Bioluminescence and microPET, respectively, of fl and tk reporter gene expression in nude mice bearing N2a tumor xenografts showed the gene expression mediated by SIRES to be 4- and 8-fold higher, respectively, than EMCV IRES. CONCLUSION: These findings support the use of SIRES bicistronic vectors for a better assessment of therapeutic gene expression based on reporter gene expression in living subjects.

Animals↗

PubMed1994

Use of the Escherichia coli beta-glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria.

A transcriptional fusion vector, designated pNZ272, based on the promoterless beta-glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed beta-glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific beta-glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.

Amino Acid Sequence↗

PubMed2002

Monitoring adenoviral DNA delivery, using a mutant herpes simplex virus type 1 thymidine kinase gene as a PET reporter gene.

Current gene therapy protocols often suffer from an inability to monitor the site, level and persistence of gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a reporter gene for in vivo imaging of reporter gene expression. The presence of the HSV1-tk reporter gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET reporter gene system, both alternative substrates and mutations in the HSV1-tk gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET reporter gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.

Adenoviridae↗

PubMed2002

Construction of transcriptional and translational lacZ gene reporter plasmids for use in Streptococcus mutans.

Reporter genes have become standard genetic tools used to evaluate either the transcriptional or the translational activity associated with genes of interest, whose products cannot be easily assayed. The lacZ gene from Escherichia coli has been used very effectively to quantify such regulated activities in many different organisms. This report describes the construction of a pair of plasmids that may be used for either transcriptional or translational lacZ gene fusions in Streptococcus mutans. The translational E. coli beta-galactosidase gene (lacZ) fusion plasmid, pALH109, as well as the transcriptional lacZ gene fusion plasmid, pALH122, have been used successfully in S. mutans to measure the activity of various PTS genes. Both plasmids employ fusions with the E. coli lacZ gene that can be easily quantified using standard O-nitrophenyl-beta-D-galactopyranoside (ONPG) based enzyme assays or the more sensitive fluorometric assays using 4-methyl-umbelliferyl beta-D-galactopyranoside (MUG) as the enzyme substrate. Currently, there has been only one other report of the use of lacZ as a gene reporter in S. mutans. The plasmids described in this paper will provide new tools and techniques for the analysis of S. mutans gene regulation. In addition, we have compiled the complete nucleotide sequences of these gene reporter plasmids.

Base Sequence↗

PubMed2001

The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis.

Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.

Animals↗

PubMed1997

Development of a bifunctional xylosidase/arabinosidase gene as a reporter gene for the gram-negative anaerobes Bacteroides and Porphyromonas, and Escherichia coli.

Members of the genera Bacteroides and Porphyromonas are common inhabitants of the human intestinal and oral microflora, and certain species are capable of causing disease states in humans. Genetic studies of these organisms are important for determining factors involved in the development of such diseases. A reporter gene for transcriptional fusions may prove useful for studies of gene regulation in these organisms. Bacteroides ovatus is a normal inhabitant of the human intestinal tract and is one of the few Bacteroides species capable of degrading xylan, a major component of fiber in the diet. A gene encoding for a bifunctional xylosidase/arabinosidase (XA) enzyme was previously cloned in our laboratory from B. ovatus V975 as part of a xylan-inducible operon. The XA gene was isolated by polymerase chain reaction and subcloned into the E. coli plasmid pBluescript II KS+. The XA gene is under transcriptional regulation in E. coli by the lac promoter, and both activities can be induced with isopropylthio-beta-galactoside (IPTG). The XA gene was subcloned into E. coli/Bacteroides shuttle vectors and introduced by conjugation into different Bacteroides species and Porphyromonas gingivalis. The results of transcriptional fusions in Bacteroides species and E. coli were evaluated. The characteristics of the XA reporter system are the low background or total lack of arabinosidase and xylosidase activities in most Bacteroides species, P. gingivalis, and E. coli, and the ease of enzymatic assays. In addition, bacterial colonies can be screened directly on agar plates by fluorescence with methylumbelliferyl derivatives as substrates for either enzymatic activity.

Artificial Gene Fusion↗

PubMed1992

[Construction and identification of vectors containing maize plasmid-like DNA S1 and reporter genes].

The reporter Gene GUS with CaMV 35S promoter was inserted into plasmid pBS[1] in reversal direction of integration, and two kinds of recombinant plasmids were obtained. These results were verified by restriction endonuclease analysis and southern hybridization. A piece of 10.8kb fragment containing NPT II reporter gene from digested pBI121.1 by BamHI and EcoRI was ligated together with 3.9 kb fragment of plasmid-like DNA S1. Then generate a recombinant plasmid pBIS5 was generated. These three vectors can be used to transform maize protoplasts. High frequency of transformation may be expected due to homology between plasmid-like DNA S1 and maize nuclear DNA.

DNA↗

PubMed2002

Variation of variable number of tandem repeat sequences in the 3'-untranslated region of primate dopamine transporter genes that affects reporter gene expression.

Genetic polymorphism has been reported in the 3'-untranslated region (3'-UTR) of the human dopamine transporter (DAT) gene and the variable number of tandem repeat (VNTR) polymorphism has been proposed to be associated with normal personal traits or psychoneurological disorders. To assess the variation of this region in nonhuman primates, we amplified the VNTR regions by the polymerase chain reaction in several species of apes and monkeys, and determined their DNA sequences. The 3'-UTR of the chimpanzee DAT gene was also polymorphic and alleles with one or two unit(s) of a 40 bp sequence were found, while all gorillas and orangutans examined had only 2-repeat allele. Cynomolgus macaques and African green monkeys shared 11- or 12-repeat and 5-repeat alleles, respectively. By performing transient transfection assay, we found that most of the VNTR sequences of nonhuman primates exhibited higher activities on reporter gene assay as compared to those of human 9-, 10- and 11-repeat sequences.

3' Untranslated Regions↗

PubMed2004

[Reporter genes transfer into human squamous cell carcinoma of larynx factor receptor mediated non-viral GE7 polypeptide delivery system with a new epidermal growth].

OBJECTIVE: To evaluate the efficiency of marker gene and reporter gene transfection into human squamous cell carcinoma of larynx implanted in nude mice with a new EGFR mediated non-viral polypeptide gene delivery system. METHODS: Labeled streptavidin biotin (LSAB) was applied to examine over-expression of epidermal growth factor receptor (EGFR) on the squamous cell carcinoma of larynx. The complex of marker gene or reporter gene and gene delivery system was prepared and subsequently inoculated with Hep 2 cells or injected into laryngeal carcinoma implanted on nude mice. RESULTS: Over-expression of EGFR was observed on inoculated Hep 2 cells and on 65% of laryngeal carcinoma specimen examined (15/23). Positive staining was located on the surface of the cells and some of them in the cytoplasma. Green fluorescence was observed on Hep 2 cells under fluorescent microscope 48 hrs after PEGFP-C1 gene transfection and became the strongest 72 hrs after transfection. The rate of transfection was 80% (78/97). Blue staining was noticed 12 hrs after beta-gal gene transfer and it became more remarkable 24 hrs after gene transfection with X-gal staining on the implanted human laryngeal carcinoma on nude mice. CONCLUSIONS: Squamous cell carcinoma of head and neck over-expressed EGFR and a non-viral GE7 polypeptide gene delivery system could transfer exogenous genes into laryngeal carcinoma cells and tissues with high efficiency and targeting both in vitro and in vivo suggesting that GE7 would be applicable as a new targeted, high efficient gene delivery system to the gene therapy of squamous cell carcinoma of head and neck.

Animals↗

PubMed1996

A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice.

We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta-galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.

3T3 Cells↗

PubMed2004

Effect of SNPs in protein kinase C zeta gene on gene expression in the reporter gene detection system.

AIM: To investigated the effects of the SNPs (rs411021, rs436045, rs427811, rs385039 and rs809912) on gene expression and further identify the susceptibility genes of type 2 diabetes. METHODS: Ten allele fragments (49 bp each) were synthesized according to the 5 SNPs mentioned above. These fragments were cloned into luciferase reporter gene vector and then transfected into HepG2 cells. The activity of the luciferase was assayed. Effects of the SNPs on RNA splicing were analyzed by bioinformatics. RESULTS: rs427811T allele and rs809912G allele enhanced the activity of the reporter gene expression. None of the 5 SNPs affected RNA splicing. CONCLUSION: SNPs in protein kinase Cz (PKCZ) gene probably play a role in the susceptibility to type 2 diabetes by affecting the expression level of the relevant genes.

Base Sequence↗

PubMed2002

In vivo analysis of glucocorticoid-induced reporter gene expression using gene gun DNA delivery.

Glucocorticoid regulates various physiological processes via the activation and repression of gene expression. The anti-inflammatory effects and the adverse effects are believed to be dependent on the repression and the activation of genes, respectively. Reporter gene assay is a useful technique to separately evaluate these two functions and has been used for in vitro screening of novel ligands for the glucocorticoid receptor (GR). We report here the application of a reporter gene assay for the in vivo determination of the GR-mediated gene activation. A reporter plasmid containing glucocorticoid response elements was introduced to abdominal mouse skin using a gene gun. Administration of prednisolone induced the expression of the reporter gene, only when the GR expression plasmid was co-transfected with the reporter plasmid. Endogenous levels of corticosterone appeared to be negligible in this protocol. The dose response for this induction was comparable to those for the decreases in thymus weight and serum corticosterone. These results suggest that gene gun-mediated skin transfection enables the in vivo reporter gene assay and that this technique can be used to predict the potency of ligands for the GR-mediated gene activation.

Animals↗

PubMed2000

Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis.

It is not known whether transcriptional suppression by de novo methylation occurs within the promoter region of the p53 gene during multistage tumorigenesis. To address this question, in vivo alterations in the CpG methylation within the rat p53 promoter region were evaluated in control, preneoplastic, and tumor tissue during tumor progression using the folate/methyl-deficient model of hepatocarcinogenesis. Alterations in CpG methylation were found to be site-specific and to vary depending on the stage of carcinogenesis. To further explore the effect of site-specific methylation on p53 promoter activity, reporter gene constructs were prepared containing specifically methylated sites within the p53 promoter region, and the transcriptional activity in cultured mammalian cells was determined in a transient transfection assay. Relative to the unmethylated construct as a positive control, single-site methylation at nucleotide (nt) -450, which occurs 216 nt upstream from the 85-nt minimal promoter region, suppressed promoter activity by 85%. In contrast, single-site methylation at nt -179, which occurs within the minimal essential promoter region, suppressed activity by only 20%. The p53 promoter constructs containing the singly methylated CpG site at nt -450 were then reevaluated for processive changes in methylation status 48 h after transfection, during maximum suppression of promoter activity. Restriction analysis with methylation-sensitive enzymes revealed that de novo methylation had occurred after transfection at previously unmethylated sites. These findings suggest that nt -450 may constitute a critical site for initiation of de novo methylation and processive spreading of methylation associated with transcriptional inactivation of the p53 gene. Furthermore, the results suggest a possible alternative mechanism for the silencing of the p53 gene in tumors that do not have p53 mutations.

Animals↗

PubMed2003

Feasibility of sodium/iodide symporter gene as a new imaging reporter gene: comparison with HSV1-tk.

Positron emission tomography (PET) imaging reporter genes, such as HSV1-tk and D(2) receptor genes, make it possible to visualise gene expression non-invasively and repetitively in vivo. However, these systems require the synthesis of complicated substrates and the availability of expensive PET equipment. Expression of the sodium/iodide symporter ( NIS) gene can be easily monitored with radioiodines and technetium-99m using a gamma camera. To evaluate the possibility of using NIS as an imaging reporter gene, we compared its characteristics with those of the conventional HSV1-tk gene. The CM cell line was made by transfecting the HSV1-tk gene into CT-26 (mouse colon carcinoma cell line). The CTN and CMN cell lines were then made by transfecting the NIS gene into CT-26 and CM. We measured the uptake of iodine-125 iodovinyldeoxyuridine ([(125)I]IVDU) and (125)I to evaluate the expression of the HSV1-tk and NIS genes, respectively. Each cell line was injected into four flank sites in Balb/c mice. The biodistribution study was performed after intravenously injecting [(125)I]IVDU and (131)I, and (131)I scintigraphy was performed for the evaluation of NIS expression. In vitro studies indicated that CTN and CMN had 40- to 79-fold and 150- to 256-fold higher uptake of (125)I than CT-26 and CM, respectively. Furthermore, CM and CMN showed 57- to 69-fold higher uptake of [(125)I]IVDU than CT-26 and CTN. NIS gene expression and (125)I accumulation were found to be directly correlated ( R(2)=0.923), as were HSV1-tk gene expression and [(125)I]IVDU accumulation ( R(2)=0.956). Calculated signal per unit NIS and HSV1-tk mRNA expression was 23,240+/-3,755 cpm and 34,039+/-5,346 cpm, respectively. In vivo study indicated that CTN and CMN had 2.3- and 5.8-fold higher uptake of (131)I than CT-26 and CM, and 1.8- and 3.5-fold higher uptake of [(125)I]IVDU than CT-26 and CTN. Scintigraphy using (131)I easily visualised CTN and CMN tumours. In conclusion, the NIS gene may be viewed as an imaging reporter gene with comparable performance to the HSV1-tk gene for monitoring target gene expression.

Animals↗

PubMed1999

Repetitive, non-invasive imaging of the dopamine D2 receptor as a reporter gene in living animals.

Reporter genes (e.g. beta-galactosidase, chloramphenicol-acetyltransferase, green fluorescent protein, luciferase) play critical roles in investigating mechanisms of gene expression in transgenic animals and in developing gene delivery systems for gene therapy. However, measuring expression of these reporter genes requires biopsy or death. We now report a procedure to image reporter gene expression repetitively and non-invasively in living animals with positron emission tomography (PET), using the dopamine type 2 receptor (D2R) as a reporter gene and 3-(2'-[18F]fluoroethyl)spiperone (FESP) as a reporter probe. We use a viral delivery system to demonstrate the ability of this PET reporter gene/PET reporter probe system to image reporter gene expression following somatic gene transfer. In mice injected intravenously with replication-deficient adenovirus carrying a D2R reporter gene, PET in vivo measures of hepatic [18F] retention are proportional to in vitro measures of hepatic FESP retention, D2R ligand binding and D2R mRNA. We use tumor-forming cells carrying a stably transfected D2R gene to demonstrate imaging of this PET reporter gene/PET reporter probe system in 'tissues'. Tumors expressing the transfected D2R reporter gene retain substantially more FESP than control tumors. The D2R/FESP reporter gene/reporter probe system should be a valuable technique to monitor, in vivo, expression from both gene therapy vectors and transgenes.

Adenoviridae↗

PubMed1998

Neuroblast pattern formation: regulatory DNA that confers the vnd/NK-2 homeobox gene pattern on a reporter gene in transgenic lines of Drosophila.

DNA fragments -0.57, -2.2, -2.9, -5.3, and -8.4 kb in length from the upstream regulatory region of the vnd/NK-2 gene were cloned in the 5'-flanking region of a beta-galactosidase (beta-gal) reporter gene in the P-element pCaSpeR-AUG-beta-gal, and the effects of the DNA on the pattern and time of expression of beta-gal were determined in transgenic embryos. Embryos from 11 lines transformed with -8.4 kb of vnd/NK-2 regulatory DNA expressed beta-gal patterns that closely resemble those of vnd/NK-2. In embryos from four lines transformed with -5.3 kb of vnd/NK-2 DNA, beta-gal was found in the normal vnd/NK-2 pattern in the nerve cord but not in part of the cephalic region. beta-Gal patterns in embryos from transgenic lines containing -0.57, -2.2, or -2.9 kb of vnd/NK-2 DNA did not resemble vnd/NK-2. Null vnd/NK-2 mutant embryos containing the homozygous P-element p[-8.4 to +0.34 beta-gal] expressed little beta-gal in contrast to siblings with a wild-type vnd/NK-2 gene. We conclude that (i) the 8.4-kb DNA fragment from the vnd/NK-2 gene contains the nucleotide sequences required to generate the normal pattern of vnd/NK-2 gene expression, sequences that may be involved in the switch between neuroblast vs. epidermoblast pathways of development, (ii) the 5'-flanking region of the vnd/NK-2 gene between -5.3 and -8. 4 kb is required for vnd/NK-2 gene expression in the most dorsoanterior part of the cephalic region, and (iii) vnd/NK-2 protein is required, directly or indirectly, for maintenance of vnd/NK-2 gene expression.

Animals↗

PubMed2004

In vivo pattern of lipopolysaccharide and anti-CD3-induced NF-kappa B activation using a novel gene-targeted enhanced GFP reporter gene mouse.

NF-kappa B is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-kappa B has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-kappa B-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-kappa B cis elements (cis-NF-kappa B(EGFP)). A gene-targeting approach was used to insert a single copy of a NF-kappa B-dependent EGFP reporter gene 5' of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-kappa B(EGFP) mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-alpha that was blocked by the NF-kappa B inhibitors BAY 11-7082 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-kappa B(EGFP) promoter. Adenoviral delivery of NF-kappa B-inducing kinase strongly induced EGFP expression in the liver of cis-NF-kappa B(EGFP) mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-kappa B(EGFP) mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-kappa B during normal and diseased states.

Animals↗