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Transient expression of chicken alpha interferon gene in lettuce.

ABSTRACT: We investigated the possibility of producing chicken alpha interferon (ChIFN-alpha) in transgenic plants. The cDNA encoding ChIFN-alpha was introduced into lettuce (Lactuca sativa L.) plants by using an agro-infiltration transient expression system. The ChIFN-alpha gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus (VSV) replication on chicken embryonic fibroblast (CEF). The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.

SUBMITTER: Song L

PROVIDER: S-EPMC2367372 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Transient expression of chicken alpha interferon gene in lettuce.

Song Li L Zhao De-gang DG Wu Yong-jun YJ Li Yi Y

Journal of Zhejiang University. Science. B 20080501 5

We investigated the possibility of producing chicken alpha interferon (ChIFN-alpha) in transgenic plants. The cDNA encoding ChIFN-alpha was introduced into lettuce (Lactuca sativa L.) plants by using an agro-infiltration transient expression system. The ChIFN-alpha gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus (VSV) replication on chick ...[more]

PMID: 18500773

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Transient Co-Expression of Bioactive Murine Interferon-Gamma and HBsAg in Tobacco and Lettuce Leaves.

Project description:BackgroundThe synchronous expression of antigen and adjuvant proteins in plant hosts presents an intriguing potential for vaccine production and the enhancement of appropriate immune responses. In this study, we examined the expression of bioactive murine interferon-gamma (mIFN-γ) along with HBsAg in tobacco and lettuce leaves aimed to further perform the analysis of immune responses in the mouse model.MethodsMonocistronic and bicistronic cassettes, carrying genes encoding mIFN-γ and HBsAg in various orders, were constructed. These cassettes were placed under the control of the 35S CaMV promoter and included the 5' leader sequence of Tobacco Ech Virus (TEV). Through Agrobacterium infiltration, the cassettes were transferred into plant leaves. The concentration of mIFN-γ in different constructs and HBsAg was tested by ELISA. Murine IFN-γ was characterized through Western blotting, and its bioactivity was evaluated by assessing the up-regulation of MHC class II in macrophages derived from mouse bone marrow.ResultsExtracts of agroinfiltrated leaves contained recombinant mIFN-γ and HBsAg proteins at about 14 unit/mg and 50 ng/mg of soluble protein, respectively. Subsequently, mIFN-γ was purified from the plant extract and its ability to up-regulate MHC class II in mouse bone marrow-derived macrophages was confirmed by immunofluorescence.ConclusionThe co-expression of recombinant HBsAg and mIFN-γ using TEV 5' leader-based cassettes in tobacco and lettuce leaves produced both proteins with active mIFN-γ in different concentrations. The attractive utility and feasibility of using plant transient co-expression systems aimed to co-delivery of vaccine antigen and appropriate cytokine to elicit immune response for different applications.

| S-EPMC10709757 | biostudies-literature

Modulation of gene expression in MHCC97 cells by interferon alpha.

Project description:AIM:To elucidate the molecular mechanisms of the inhibitory effects of IFN-alpha on tumor growth and metastasis in MHCC97 xenografts. METHODS:Three thousand international units per milliliter of IFN-alpha-treated and -untreated MHCC97 cells were enrolled for gene expression analysis using cDNA microarray. The mRNA levels of several differentially expressed genes in cDNA microarray were further identified by Northern blot and RT-PCR. RESULTS:A total of 190 differentially expressed genes including 151 IFN-alpha-repressed and 39-stimulated genes or expressed sequence tags from 8,464 known human genes were found to be regulated by IFN-alpha in MHCC97. With a few exceptions, mRNA levels of the selected genes in RT-PCR and Northern blot were in good agreement with those in cDNA microarray. CONCLUSION:IFN-alpha might exert its complicated anti-tumor effects on MHCC97 xenografts by regulating the expression of functional genes involved in cell metabolism, proliferation, morphogenesis, angiogenesis, and signaling.

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The differential antiviral activities of chicken interferon ? (ChIFN-?) and ChIFN-? are related to distinct interferon-stimulated gene expression.

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Pegylated IFN-α regulates hepatic gene expression through transient Jak/STAT activation.

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Interferon-alpha drives monocyte gene expression in chronic unsuppressed HIV-1 infection.

Project description:OBJECTIVES:HIV-1 infection dysregulates the innate immune system and alters leukocyte-gene expression. The objectives were two fold: to characterize the impact of HIV-1 infection on peripheral monocyte gene expression and to identify the predominant factor(s) responsible for altered gene expression. DESIGN AND METHODS:In a cross-sectional study (n = 55), CD14 monocytes were isolated from 11 HIV-1 seronegative controls, 22 HIV-1 seropositive individuals with low-viral loads (LVL) and 22 HIV-1 seropositive individuals with high-viral loads (HVL). Monocyte gene expression data were collected for control, LVL and HVL individuals using high-density microarrays. We evaluated three HIV-1 disease-related peripheral factors, interferon (IFN)-alpha, IFN-gamma and lipopolysaccharide (LPS) as candidates causing monocyte dysregulation, by comparing gene expression profiles between study individuals and monocytes treated with these factors in vitro. Plasma from HIV-1 positive individuals was quantified for LPS and soluble CD14. RESULTS:Monocytes from HIV-1-infected individuals with viral loads above 10,000 RNA copies/ml (HVL) displayed an activated phenotype. Characterization of gene expression revealed an ongoing immune response to viral infection including inflammation and chemotaxis. Gene expression analysis of in-vitro-treated HIV-1 seronegative monocytes with IFN-alpha, IFN-gamma or LPS demonstrated that IFN-alpha most accurately recapitulated the HIV-1 HVL profile. No LPS-induced gene expression signature was detected even in HIV-1 individuals with the highest LPS and sCD14 levels. CONCLUSION:Monocyte gene expression in individuals with HIV-1 viremia is predominantly due to IFN-alpha, whereas individuals with LVL have a nonactivated phenotype. In monocytes, there was no discernible expression profile linked to LPS exposure.

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Chickspress: a resource for chicken gene expression

Project description:Red Jungle Fowl (male and female) tissues were analyzed using LC-MS/MS. Tissues analyzed were: adipose, adrenal gland, breast muscle, cerebellum, cerebrum, gonad, heart, hypothalamus, kidney, liver, lung, pancreas, proventriculus, sciatic nerve, speen. Samples were analyzed using an LTQ Velos Pro mass spectrometer. Xtandem was used to perform spectrum searches. Databases included are NCB refseq, Ensembl, and 6-frame translation of the chicken genome.

2016-11-07 | MSV000080320 | MassIVE

CTCF demarcates chicken embryonic α-globin gene autonomous silencing and contributes to adult stage-specific gene expression.

Project description:Genomic loci composed of more than one gene are frequently subjected to differential gene expression, with the chicken α-globin domain being a clear example. In the present study we aim to understand the globin switching mechanisms responsible for the epigenetic silencing of the embryonic π gene and the transcriptional activation of the adult α(D) and α(A) genes at the genomic domain level. In early stages, we describe a physical contact between the embryonic π gene and the distal 3' enhancer that is lost later during development. We show that such a level of regulation is achieved through the establishment of a DNA hypermethylation sub-domain that includes the embryonic gene and the adjacent genomic sequences. The multifunctional CCCTCC-binding factor (CTCF), which is located upstream of the α(D) gene promoter, delimits this sub-domain and creates a transition between the inactive sub-domain and the active sub-domain, which includes the adult α(D) gene. In avian-transformed erythroblast HD3 cells that are induced to differentiate, we found active DNA demethylation of the adult α(D) promoter, coincident with the incorporation of 5-hydroxymethylcytosine (5hmC) and concomitant with adult gene transcriptional activation. These results suggest that autonomous silencing of the embryonic π gene is needed to facilitate an optimal topological conformation of the domain. This model proposes that CTCF is contributing to a specific chromatin configuration that is necessary for differential α-globin gene expression during development.

| S-EPMC3883786 | biostudies-literature

Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α).

Project description:Viruses that infect birds pose major threats-to the global supply of chicken, the major, universally-acceptable meat, and as zoonotic agents (e.g. avian influenza viruses H5N1 and H7N9). Controlling these viruses in birds as well as understanding their emergence into, and transmission amongst, humans will require considerable ingenuity and understanding of how different species defend themselves. The type I interferon-coordinated response constitutes the major antiviral innate defence. Although interferon was discovered in chicken cells, details of the response, particularly the identity of hundreds of stimulated genes, are far better described in mammals. Viruses induce interferon-stimulated genes but they also regulate the expression of many hundreds of cellular metabolic and structural genes to facilitate their replication. This study focusses on the potentially anti-viral genes by identifying those induced just by interferon in primary chick embryo fibroblasts. Three transcriptomic technologies were exploited: RNA-seq, a classical 3'-biased chicken microarray and a high density, "sense target", whole transcriptome chicken microarray, with each recognising 120-150 regulated genes (curated for duplication and incorrect assignment of some microarray probesets). Overall, the results are considered robust because 128 of the compiled, curated list of 193 regulated genes were detected by two, or more, of the technologies.

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Gene cloning, sequencing, expression and biological activity of giant panda (Ailuropoda melanoleuca) interferon-alpha.

Project description:The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. In mammals, multiple subtypes of interferon-alpha (IFN-alpha) exist, most of which possess antiviral activity. Little is known about giant panda IFN-alpha genes and the role they may play in giant panda immunological responses to viruses. We have cloned genes encoding 12 giant panda IFN-alpha (AmIFN-alpha or AmIFNA) subtypes that share from 90 to 99% amino acid sequence identity. AmIFN-alpha12 has one additional amino acid at position 57, which is not present in other subtypes. Sequence identity of the AmIFN-alpha proteins encoded by the 12 genes compared to human IFN-alpha2 is approximately 58%. Unlike most of the human subtypes, each of the 12 giant panda IFN sequences has an N-glycosylation recognition site. Expression of all 12 AmIFN-alpha subtypes in 293 cells was confirmed by SDS-PAGE and Western blotting analysis. The antiviral activity and antiproliferative activity of each AmIFN-alpha subtype produced in transiently transfected 293 cell cultures were tested in vitro. All AmIFN-alpha subtypes were found to be stable at pH 2 or 65 degrees C and to exhibit antiviral activity. Some IFN subtypes (AmIFN-alpha8 and AmIFN-alpha4) showed higher biological activity levels than others, whereas AmIFN-alpha11 exhibited lower activity. AmIFN-alpha had various antiproliferative activities to different target cells. To B16 cells, AmIFN-alpha3, AmIFN-alpha4, AmIFN-alpha8 had the highest activities, while to K562 cells, AmIFN-alpha3, AmIFN-alpha7, AmIFN-alpha10 had the highest activities. The various IFN-alpha subtypes displayed a good correlation between their antiviral and antiproliferative potencies.

| S-EPMC7112545 | biostudies-literature

Gene expression profiles in an hepatitis B virus transfected hepatoblastoma cell line and differentially regulated gene expression by interferon-alpha.

Project description:AimTo study interactions between hepatitis B virus (HBV) and interferon-alpha in liver- derived cells.MethodsmRNAs were separately isolated from an HBV-transfected cell line (HepG(2)2.2.15) and its parental cell line (HepG(2)) pre- and post-interferon-alpha (IFN-alpha) treatment at 6, 24 and 48 h, followed by hybridization with a cDNA microarray filter dotted with 14 000 human genes. After hybridization and scanning of the arrays, the data were analyzed using ArrayGauge software. The microarray data were further verified by Northern blot analysis.ResultsCompared to HepG(2) cells, 14 genes with known functions were down-regulated 3 to 12- magnitudes, while 7 genes were up-regulated 3-13 magnitudes in HepG(2)2.2.15 cells prior to IFN-alpha treatment. After interferon-alpha treatment, the expression of four genes (vascular endothelial growth factor, tyrosine phosphate 1E, serine protein with IGF-binding motif and one gene of clathrin light chain) in HepG(2)2.2.15 were up-regulated, while one gene encoding a GTP-binding protein, two genes of interferon-induced kinases and two proto-oncogenes were further down- regulated. Interestingly, under IFN-alpha treatment, a number of differentially regulated genes were new ESTs or genes with unknown functions.ConclusionThe up-regulated genes in HepG(2)2.2.15 cell line suggested that under IFN-alpha treatment, these repressed cellular genes in HBV infected hepatocytes could be partially restored, while the down- regulated genes were most likely the cellular genes which could not be restored under interferon treatment. These down-regulated genes identified by microarray analysis could serve as new targets for anti-HBV drug development or for novel therapies.

| S-EPMC4572260 | biostudies-literature

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