<i>Porphyromonas gingivalis</i>-Stimulated TACE Activation for TGF-<i>α</i>Ectodomain Shedding and EGFR Transactivation in Salivary Gland Cells Requires Rac1-Dependent p38 MAPK Membrane Localization

Oral mucosal inflammatory responses to P. gingivalis and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in proinflammatory cytokine production, up-regu- lation in mitogen-activated protein kinase (MAPK) cascade, and the induction in epidermal growth factor receptor (EGFR) activation. In this study, we report that stimulation of salivary gland acinar cells with P. gingivalis LPS leads to p38 MAPK-dependent release of soluble TGF-α ligand and the increase in EGFR phosphorylation. Further, we show that the LPS-induced TGF-α shedding and EGFR transactivation involve the activation of membrane-associated metalloprotease, TACE also known as ADAM17, through phosphorylation by p38 MAPK, and require Rac1 participation. Moreover, we demonstrate that blocking the Rac1 activation leads to the suppression in the membrane translocation of Rac1 as well as p38, thus indicating that the LPS-elicited p38 membrane recruitment for TACE phosphorylation requires colocalization with Rac1. Hence, our findings imply that Rac1 membrane translocation serves as an essential platform for the localization of p38 with TACE, TGF-α ectodomain shedding, and the EGFR activation.

Hydrogen sulfide-linked persulfidation of ABI4 controls ABA responses through the transactivation of MAPKKK18 in Arabidopsis

Hydrogen sulfide(H2S)is a signaling molecule that regulates plant hormone and stress responses.The phytohormone abscisic acid(ABA)plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1(DES1)and the production of H2S in guard cells.However,it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals.In this study,we discovered that ABSCISIC ACID INSENSITIVE 4(ABI4)acts downstream of DES1 in the control of ABA responses in Arabidopsis.ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA,and loss of DES1 function impairs this process.Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination,seedling establishment,and stomatal closure,which are abolished in the ABI4Cys250Ala mutated variant.Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA.Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18(MAPKKK18),which propagates the MAPK signaling cascade induced by ABA.Furthermore,the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18,and ABI4 can bind the DES1 promoter,forming a regulatory loop.Taken together,these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.

EYA4 inhibits hepatocellular carcinoma growth and invasion by suppressing NF-κB-dependent RAP1 transactivation

Background:Our previous studies demonstrated that eyes absent homolog 4(EYA4),a member of the eye devel-opment-related EYA family in Drosophila,is frequently methylated and silenced in hepatocellular carcinoma(HCC)specimens and associated with shorter survival.The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.Methods:Stable EYA4-expressing plasmid(pEYA4)transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5(PLC)were established.Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice.Tissue samples were obtained from 75 pathologically diagnosed HCC patients.Quantitative real-time polymerase chain reaction,Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines,xenografts and clinical specimens.The cell proliferation,colony formation,invasiveness and tumor formation of stable transfectants were studied.A gene expression microarray was utilized to screen genes regulated by EYA4 expression.The effect of EYA4 on nuclear factor-κB(NF-κB)/RAS-related protein 1(RAP1)signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid(Flag-RAP1A),functional studies,chromatin immunoprecipitation,immunofluorescence staining and cellular ubiquitination assay.Results:The restoration of EYA4 expression in HCC cell lines suppressed cell proliferation,inhibited clonogenic outgrowth,reduced cell invasion and restrained xenograft tumor growth,and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro.Activation of NF-κB with tumor necrosis factor-α(TNF-α)increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression.The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation.EYA4 antagonized the TNF-α-induced phosphoryla-tion and ubiquitination of inhibitor of NF-κBα(IκBα)as well as the nuclear translocation and transactivation of p65,resulting in repressed NF-κB activity and RAP1 expression.Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity.In addition,EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.Conclusion:Our findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor sup-pressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.

Activation of GABAB receptors protects cerebellar granule neurons from apoptosis via IGF-I receptor transactivation

γ-amidobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and mediates fast synaptic inhibition through GABAA and GABAC

Subcloning and Expression of Human Papillomavirus Type 16 E2 Gene in E.Coli

By using molccular doning technique,the E2 gene of human papillomavirus type 16 wasexpressed in E.coli.The 3.2 kb fragment containing the E2 gene was cut out from HPV16 genomeand blunted with nuelease S1.The plasmid pBD2 DNA was linearized with Hind Ⅲ and bluntedwith nuclasc S1 too.Afler ligation,thc ligsted DNA was used to transform E.coli BMH 71-18.The positive colonies were screened by in situ hybridization technique,and proceedcd to DNA analy-sis and proton analysis.The purified expressed protein was used to run immunoclctrophoreesis withantiserum against pBD2.We concktudcd that thc expressed protcin was a fusion protein ofbeta-galae-sidasc-E2 protein.

Identification of genes upregulated by recombinant interferon-alpha in HepG2 cells by suppressive subtractive hybridization analysis

BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully, rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.

A Toxicological Assessment of Endocrine Disrupting Chemicals Found in the BMW (Border, Midland and Western) Region of Ireland

A battery of tests was established to determine the oestrogenic, mutagenic and genotoxic potential of two categories of endocrine disrupting chemicals (EDCs), phthalates and alkylphenols. Diisononylphthalate (DINP), diethylhexylphthalate (DEHP), dibutylphthalate (DBP), diisododecylphthalate (DIDP) and 4-nonylphenol (4-NP) were oestrogenic in the yeast estrogen screen (YES) assay and potently oestrogenic in the MVLN and E-SCREEN assays at environmentally relevant concentrations. DINP and 4-NP were mutagenic in the Ames assay and also induced significant levels of unscheduled DNA synthesis and DNA strand breakage. Significant induction in the percentage of cells containing micronuclei was observed after treatment with DINP, DEHP and 4-NP. In addition, sewage effluents from sewage treatment plants (STPs) in the Border, Midlands and Western (BMW) region of Ireland were significantly oestrogenic in the YES assay. Moreover, analysis of levels of phthalates and alkylphenol identified in Irish rivers receiving treated effluent showed potent oestrogenicity in the YES assay. The proliferative and genotoxic ability of the phthalates and alkylphenol, and the oestrogenicity of the treated effluents reported here, is significant as these EDCs and EDCs within the effluent may play a role in the etiology of human abnormalities.

In Vitro Biological Activity of Anti-C Ⅱ TA Hammerhead Ribozyme——A Novel Approach for Autoimmune Diseases

This study investigated the feasibility of using an hammerhead ribozyme against C Ⅱ TA, a major regulator of MHC Ⅱ antigens, to repress the expression of MHC Ⅱ molecules on Hela cells. A hammerhead ribozyme (Rz464) specific to 463-465 GUC triplet of C Ⅱ TA and its target gene were transcribed, then mixed up and incubated in vitro . The cleavage products were analyzed by PAGE and silver staining. Rz464 was then inserted into the pIRES2 EGFP vector (pRz464). Stable transfectants of Hela with pRz464 were tested for class Ⅱ MHC induction by recombinant human interferon gamma (IFN γ). mRNA of C Ⅱ TA was measured by RT PCR. Our results showed that Rz464 could exclusively cleave C Ⅱ TA RNA. When induced with IFN γ, the expression of HLA DR, DP, DQ on pRz464 + Hela was induced, and the mRNA content of C Ⅱ TA decreased too. It is concluded that Rz464 could inhibit C Ⅱ TA and thus the family of genes was regulated by C Ⅱ TA:MHC Ⅱ molecules. These results provided insight into the future application of Rz464 as a new nucleic acid drug against auto immune diseases.

Establishment of a tetracycline-off and heat shock-on gene expression system in tobacco

The tetracycline （Tet）-off gene expression regulation system based on the TetR-VP16/Top10 construct has not been widely utilized in plants, for its highly expressed TetR-VP16 activator is toxic to some plants and repeatedly replenishing tetracycline to turn off the constitutively active system is a tedious process. To solve these problems, a Tet-off and heat shock （HS）-on gene expression regulation system was constructed in this study. This system is composed of a chimeric transactivator gene TetR-HSF that is derived from a Tet repressor （TetR） and a HS transcription factor （HSF） controlled by a HS promoter HSP70m, and a Tet operator containing hybrid promoter, Om35S, that drives expression of the β-glucuronidase （GUS） gene. The resultant system yields a GUS expression pattern similar to that of the HSP70m promoter under inducing temperatures and at 35 and 40℃ drives GUS expression to a similar level as the Cauliflower mosaic virus （CaMV） 35S promoter. Further examination revealed that the TetR-HSF and GUS genes were induced by HS, reaching peak expression after 1 and 6 h treatment, respectively, and the HS induction of the expression system could be inhibited by Tet. This system will provide a useful tool for transgenic studies of plants in the laboratory and in the field, including transgene function analysis, agronomic trait improvement, biopharmaceutical protein production and others.

The enhancement of astrocytic-derived monocyte chemoattractant protein-1 induced by the interaction of opiate and HIV tat in HIV-associated dementia

HIV-associated dementia(HAD)is a public health problem and is particularly prevalent in drug abusers.The neuropathogenesis of human immunodeficiency virus(HIV)infection involves a complex cascade of inflammatory events,including monocyte/macrophage infiltration in the brain,glial immune activation and release of neurotoxic substances.In these events,astrocytic-derived monocyte chemoattractant protein-1(MCP-1)plays an important role,whose release is elevated by HIV transactivator of transcription(HIV tat)and could be further elevated by opiates.This review will also consider some critical factors and events in MCP-1 enhancement induced by the interactions of opiate and HIV tat,including the mediating role of mu opioid receptor(MOR)and CCR2 as well as the possible signal transduction pathways within the cells.Finally,it will make some future perspectives on the exact pathways,new receptors and target cells,and the vulnerability to neurodegeneration with HIV and opiates.

Expression of CⅡTA Gene in Five Human Malignant Hematological Cell Lines and Its Significance

The role of MHC class Ⅱ transactivator (CⅡTA) in constitutive or IFN-γ inducible expression of HLA molecules in human malignant hematological cell lines was investigated. The expression of HLA molecules and CⅡTA protein was detected by Western blot, immunohistochemistry and flow cytometry. The expression of CⅡTA gene was determined by RT-PCR. The capability of peripheral blood T cell reaction stimulated by tumor cells was monitored by mixed lymphocyte reaction. It was found that the HLAⅡ-positive tumor cells expressed the CⅡ TA quite well, and the expression of HLAⅠ+Ⅱ was increased in the tumor cells with constitutive or inducible expression of CⅡ TA after induced by IFN-γ. The tumor cells which did not express CⅡ TA after induced by IFN- γ were not response to the expression of HLAⅡ promoted by IFN- γ. It suggests a correlation between the inability of some malignant hematological cell lines in response to IFN-γ for HLA expression and the deficiency in the inducible expression of CⅡTA, indicating CⅡ TA might take part in the regulation of HLA Ⅰ+Ⅱ expression in the tumor cells, which might play an important role in tumor immunologic escape.

TNF-α Induces Transient Resistance to Fas-Induced Apoptosis in Eosinophilic Acute Myeloid Leukemia Cells

Tumor necrosis factor α （TNF-α） has been recognized as an activator of nuclear factor kB （NF-kB）, a factor implicated in the protection of many cell types from apoptosis. We and others have presented evidence to suggest that Fas-induced apoptosis may be an important aspect of the resolution of inflammation, and that delayed resolution of inflammation may be directly associated with NF-kB-dependent resistance to Fas. Because TNF-α activates NF-kB in many cell types including inflammatory cells such as eosinophils, we examined effects of TNF-α signaling on the Fas-mediated killing of an eosinophilic cell line AML14. While agonist anti-Fas （CHII） treatment induced apoptosis in AML14 cells, no significant cell death occurred in response to TNF-α alone. Electrophoretic mobility shift assay （EMSA） revealed that TNF-α induced NF-kB transactivation in AMLI4 cells in a time- and dose-dependent fashion, and subsequent supershift assays indicated that the translocated NF-kB was the heterodimer p65 （RelA）/p50. Pre-treatment of cells with TNF-α dramatically decreased the CHll-induced cell death in a transient fashion, accompanied by suppression of activation of caspase-8 and caspase-3 activation. Inhibition of NF-kB transactivation by inhibitors, BAY 11-7085 and parthenolide, reversed the suppression of Fas-mediated apoptosis by TNF-α. Furthermore, TNF-α up-regulated X-linked inhibitor of apoptosis protein （XIAP） transiently and XIAP levels were correlated with the temporal pattern of TNF-α protection against Fas-mediated apoptosis. This finding suggested that TNF-α may contribute to the prolonged survival of inflammatory cells by suppression of Fas-mediated apoptosis, the process involved with NF-kB transactivation, anti-apoptotic XIAP up-regulation and caspase suppression. Cellular ＆ Molecular Immunology. 2007;4（1）：43-52.

Role of vitamin D receptor in the regulation of CYP3A gene expression

Vitamin D3(VD3)is a multifunctional nutrient which can be either synthesized or absorbed from the diet.It plays a pivotal role in systemic calcium and phosphate homeostasis,as well as in various physiological and pathological processes.VD3 is converted to the active form,1α,25-dihydroxy vitamin D3(1,25-D3),by cytochrome P4502R1(CYP2R1)/CYP27A1 and CYP27B1 sequentially,and deactivated by multiple enzymes including CYP3A4.On the other hand,1,25-D3 is capable of activating the transcription of CYP3A genes in humans,mice and rats.The vitamin D receptor(VDR)-mediated transactivation of human CYP3A4 and CYP3A5 resembles that known for pregnane X receptor(PXR).Activated VDR forms a heterodimer with retinoid X receptorα(RXRα),recruits co-activators,translocates to the cell nucleus,binds to the specific vitamin D responsive elements(VDRE),and activates the gene transcription.In mice,intestinal Cyp3a11 mRNA levels,but not those of hepatic CYP3As,were induced by in vivo administration of VDR and PXR agonists.In rats,intestinal Cyp3a1 and Cyp3a2mRNAs were induced by 1,25-D3 or lithocholic acid(LCA),whereas hepatic Cyp3a2,but not Cyp3a1and Cyp3a9,was modulated to 1,25-D3 treatment.In general,the VDR-mediated regulation of CYP3A presents species and organ specificity.

Rice GLUTATHIONE PEROXIDASE1-mediated oxidation of bZIP68 positively regulates ABA-independent osmotic stress signaling

Osmotic stress caused by drought and high salinity is a significant environmental threat that limits plant growth and agricultural yield. Redox regulation plays an important role in plant stress responses, but the mechanisms by which plants perceive and transduce redox signals are still underexplored. Here, we report a critical function for the thiol peroxidase GPX1 in osmotic stress response in rice, where it serves as a redox sensor and transducer. GPX1 is quickly oxidized upon exposure to osmotic stress and forms an intramolecular disulfide bond, which is required for the activation of bZIP68, a VRE-like basic leucine zipper (bZIP) transcription factor involved in the ABA-independent osmotic stress response pathway. The disulfide exchange between GPX1 and bZIP68 induces homo-tetramerization of bZIP68 and thus positively regulates osmotic stress response by regulating osmotic-responsive gene expression. Furthermore, we discovered that the nuclear translocation of GPX1 is regulated by its acetylation under osmotic stress. Taken together, our findings not only uncover the redox regulation of the GPX1-bZIP68 module during osmotic stress but also highlight the coordination of protein acetylation and redox signaling in plant osmotic stress responses.

Steroid hormone receptors and prostate cancer： role of structural dynamics in therapeutic targeting

Steroid hormone receptors （SHRs） act in cell type- and gene-specific manner through interactions with coregulatory proteins to regulate numerous physiological and pathological processes at the level of gene regulation. Binding of steroid receptor modulator （SRM） ligand leads to allosteric changes in SHR to exert positive or negative effects on the expression of target genes. Due, in part, to the fact that current SRMs generally target ligand binding domain （LBD）/AF2 and neglect intrinsically disordered （ID） N-terminal domain （NTD）/AF1, clinically relevant SRMs lack selectivity and are also prone to the development of resistance over time. Therefore, to maximize the efficacy of SHR-based therapeutics, the possibility of developing unique modulators that act to control AF1 activity must be considered. Recent studies targeting androgen receptor＇s （AR＇s） ID AF1 domain for the castration-resistant prostate cancer has provided the possibility of therapeutically targeting ID NTD/AF1 surfaces by allosteric modulations to achieve desired effects. In this review article, we discuss how inter- and intra- molecular allosteric regulations controlled by AR＇s structural flexibility and dynamics particularly the ID NTD/AF1 is an emerging area of investigation, which could be exploited for drug development and therapeutic targeting of prostate cancer.

Characterization of a novel missense mutation on murine Pax3 through ENU mutagenesis

N-ethyl-N-nitrosourea （ENU） mutagenesis has led to the elucidation of several regulator genes for melanocyte and skin development. Here we characterized a mutant from ENU mutagenesis with similar phenotype as that of Splotch mutant, including exencephaly, spina bifida and abnormal limbs in homozygotes as well as white belly spotting and occasionally loop-tail in heterozygotes. This novel mutant was named as SpxG. Through genome-wide linkage analysis in backcross progenies with microsatellite markers, the SpxG was confined to a region between DIMIT415 and DIMIT7 on chromosome 1, where notable Pax3 gene was located. Direct sequencing revealed that SpxG carried a nucleotide A894G missense transition in exon 6 of Pax3 gene that resulted in Asn to Asp substitution at amino acid 269 within the highly-conserved homeodomain （HD） DNA recognition module, which was the first point mutation found in this domain in mice. This N269D mutation impaired the transactivation capacity of Pax3 protein, but exerted no effect on Pax3 protein translation. The characterization of the new mutation expanded our understanding the transactivation and DNA-binding structure of Pax3 protein.

Reprogramming cells with synthetic proteins

Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors （TFs） has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to ＂read＂ genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongeneticaUy by using drug.like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore; the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8

Kaposi’s sarcoma-associated herpesvirus(KSHV)is γ-2 herpesvirus with latency and lytic replication stages in its life-cycle.The viral replication and transcription activator(RTA)is the key protein for triggering KSHV lytic gene expression and replication from latency.In this review,we will discuss the gene expression program in KSHV lytic replication and latency,the regulation of the RTA expression,the RTA protein and the mechanisms that RTA utilizes to transactivate its target genes.We will focus on the RTA-mediated transactivation mechanisms,including DNA-binding,interacting with cellular co-factors and promoting repressor degradation.

Lnc-RP5 Regulates the mi R-129-5p/Notch1/PFV Internal Promoter Axis to Promote the Expression of the Prototype Foamy Virus Transactivator Tas

Prototype foamy virus(PFV)is a unique retrovirus that infects animals and humans and does not cause clinical symptoms.Long noncoding RNAs(lncRNAs)are believed to exert multiple regulatory functions during viral infections.Previously,we utilized RNA sequencing(RNA-seq)to characterize and identify the lncRNA lnc-RP5-1086 D14.3.1-1:1(lnc-RP5),which is markedly decreased in PFV-infected cells.However,little is known about the function of lnc-RP5 during PFV infection.In this study,we identified lnc-RP5 as a regulator of the PFV transcriptional transactivator(Tas).Lnc-RP5 enhanced the activity of the PFV internal promoter(IP).The expression of PFV Tas was found to be promoted by lnc-RP5.Moreover,mi R-129-5 p was found to be involved in the lnc-RP5-mediated promotion of PFV IP activity,while the Notch1 protein suppressed the activity of PFV IP and the expression of Tas.Our results demonstrate that lnc-RP5 promotes the expression of PFV Tas through the miR-129-5 p/Notch1/PFV IP axis.This work provides evidence that host lnc RNAs can manipulate PFV replication by employing mi RNAs and proteins during an early viral infection.

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

Conditional expression of a target gene during zebrafish development is a powerful approach to elucidate gene functions. The tetracycline-controlled systems have been successfully used in the modulation of gene expression in mammalian cells, but few lines of zebrafish carrying these systems are currently available. In this study, we had generated a stable transgenic zebrafish line that ubiquitously expressed the second-generation of reverse Tet transactivator （rtTA-M2）. Southern blotting analysis and high-throughput genome sequencing verifed that a single copy of rtTA-M2 gene had stably integrated into the zebrafish genome. After induction with doxycycline （Dox）, a strong green fluorescent protein （GFP） was seen in rtTA-transgenic eggs injected with pTRE--EGFP plasmids. The fluorescent signal gradually decreased after the withdrawal of Dox and disappeared. However, leaky expression of GFP was undetectable before Dox- induction. Additionally, transgenic embryos expressing rtTA-M2 exhibited no obvious defects in morphological phenotypes, hatching behavior and expression patterns of developmental marker genes, suggesting that rtTA-M2 had little effect on the development of transgenic zebrafish. Moreover, expressed Dickkopf-1 （DKK1） in pTRE-DKKl-injected embryos led to alterations in the expression of marker genes associated with Wnt signaling. Thus, this rtTA-transgenic zebrafish can be utilized to dissect functions of genes in a temporal manner.