Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.

Neoplastic Transformation of T Lymphocytes through Transgenic Expression of a Virus Host Modification Protein

Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4(+)CD8(+)CD69(-) lymphoma. The CD4(+)CD8(+)CD69(-) cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4(+)CD8(+)CD69(-) thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4(+)CD8(+) CD69(-) thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-)oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas.

GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon

Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, DeltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551D) K18-GFP-CFTR+/-, designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.

A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse

The c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in expression of the gene in trophoblasts. We show that in mice, c-fms transcripts in trophoblasts initiate from multiple points within the 2-kilobase (kb) region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5' flanking sequence and the down-stream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP highlighted the large numbers of c-fms-positive macrophages, including those that originate from the yolk sac. In adult mice, EGFP location Was consistent with known F4/80-positive macrophage populations, including Langerhans cells of the skin, and permitted convenient sorting of isolated tissue macrophages from disaggregated tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either all of intron 2 or a conserved enhancer element FIRE (the Fms intronic regulatory element) was removed. We have therefore defined the elements required to generate myeloid- and trophoblast-specific transgenes as well as a model system for the study of mononuclear phagocyte development and function. (C) 2003 by The American Society of Hematology.

HIV LTR-dependent expression of Bax selectively induces apoptosis in Tat-positive cells

HIV integrates into the host cell genome where it persists for the life of the cell. One approach to reducing viral burden is to selectively eliminate cells containing integrated provirus early following infection. We have used the HIV LTR promoter to selectively express transgenes in human cells positive for the HIV transactivator protein Tat. Transient transfection of Jurkat cells, or Jurkat cells stably expressing Tat (Jurkat-Tat), with a LTR construct containing luciferase reporter gene resulted in a 37-fold increase in gene expression when Tat was present. We have demonstrated that when pro-apoptotic Bax was used as the transgene, cytotoxicity was seen only in the Jurkat-Tat cells. Annexin-V staining indicated that Bax induced cell death by apoptosis. In mixed populations of Jurkat and Jurkat-Tat cells, the LTR-Bax construct was selectively cytotoxic to the Tat-positive cells. These results suggest that Bax under the control of the HIV LTR can be used to destroy cells harbouring HIV without affecting uninfected cells. (C) 2004 Published by Elsevier Inc.

Biological response of broiler chickens fed peas (Pisum sativum L.) expressing the bean (Phaseolus vulgaris L.) alpha-amylase inhibitor transgene

The nutritive value of transgenic peas expressing an a-amylase inhibitor (alpha-Ail) was evaluated with broiler chickens. The effects of feeding transgenic peas on the development of visceral organs associated with digestion and nutrient absorption were also examined. The chemical composition of the conventional and the transgenic peas used in this study were similar. In the two feeding trials, that were conducted normal and transgenic peas were incorporated into a maize-soybean diet at concentrations up to 500 g kg(-1). The diets were balanced to contain similar levels of apparent metabolisable energy (AME) and amino acids. In the first trial, the birds were fed the diets from 3 to 17days post-hatching and with levels of transgenic peas at 250 g kg(-1) or greater there was a significant reduction in body weight but an increase in feed intake resulting in deceased feed conversion efficiency. In the second trial, in which the birds were fed diets containing 300 g kg(-1) transgenic peas until 40 days of age, growth performance was significantly reduced. It was also demonstrated that the ileal starch digestibility coefficient (0.80 vs 0.42) was significantly reduced in the birds fed transgenic peas. Determination of AME and ileal digestibility of amino acids in 5-week-old broilers demonstrated a significant reduction in AME (12.12 vs 5.08 MJ kg(-1) DM) in the birds fed the transgenic peas. The AME value recorded for transgenic peas reflected the lower starch digestibility of this line. Real digestion of protein and amino acids was unaffected by treatment. Expression of a-Ail in peas did not appear to affect bird health or the utilisation of dietary protein. However, the significant reduction in ileal digestion of starch in transgenic peas does reduce the utility of this feedstuff in monogastric diets where efficient energy utilisation is required. (c) 2006 Society of Chemical Industry.

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.

Over-Expression of Endothelin 3 Sensitizes Mice to Uv-Induced Melanoma Metastasis

Melanomagenesis is influenced by environmental and genetic factors. In normal cells, ultraviolet (UV) induced photoproducts are successfully repaired by the nucleotide excision repair (NER) pathway. Mice carrying mutations in the xeroderma pigmentosum (Xp) complementation group of genes (Xpa-Xpg) lack the NER pathway and are therefore highly sensitive to UV light; however, they do not develop melanoma after UV exposure. In humans, the Endothelin 3 signaling pathway has been linked to melanoma progression and its metastatic potential. Transgenic mice that over-express Edn3 under the control of the Keratin 5 promoter (K5-Edn3) and exhibit a hyperpigmentation phenotype, were crossed with Xp deficient mice. Because melanoma is highly metastatic and many primary malignancies spread via the lymphatic system, analyzing the lymph nodes may serve useful in assessing the possible spread of tumor cells to other tissues. This study aimed to determine whether the over-expression of Edn3 is sufficient to lead to melanoma metastasis to the lymph nodes. Mice were exposed to UV radiation and analyzed for the presence of skin lesions. Mice presenting skin lesions were sacrificed and the nearest lymph nodes were excised and examined for the presence of metastasis. Mice with melanoma skin lesions presented enlarged and hyperpigmented lymph nodes. Diagnosis of melanoma was established by immunostaining with melanocyte and melanoma cell markers, and while UV radiation caused the development of skin lesions in both K5-Edn3 transgenic and control mice, only those mice carrying the K5-Edn3 transgene were found to develop melanoma metastasis to the lymph nodes. These results indicate that over-expression of Edn3 is sufficient to lead to lymph node metastasis in mice exposed to at least one dose of UV radiation.

Growth dependent silencing and resetting of DGA1 transgene in Nannochloropsis salina

Here we report recombinant expression and activity of the Saccharomyces cerevisiae type 2 diacylglycerol acyltransferase DGA1 functioning in parallel with the native Nannochloropsis salina genes. Expression of DGA1 shifted the chain length distribution of fatty acids produced and reflected an oleoyl-CoA substrate preference. Effect on the total FAME content was moderate and elevated by a maximum of 38%. Expression of the DGA1 transgene varied throughout the culture life cycle and evidence of growth dependent environmental silencing of the transgene was observed. This is to our knowledge the first example of silencing and subsequent resetting in a transgenic microalga. Results from this study add valuable insights into the efficacy of algal genetic engineering and use of these microorganisms as bio-platforms for chemical manufacture.

Growth dependent silencing and resetting of DGA1 transgene in Nannochloropsis salina

Here we report recombinant expression and activity of the Saccharomyces cerevisiae type 2 diacylglycerol acyltransferase DGA1 functioning in parallel with the native Nannochloropsis salina genes. Expression of DGA1 shifted the chain length distribution of fatty acids produced and reflected an oleoyl-CoA substrate preference. Effect on the total FAME content was moderate and elevated by a maximum of 38%. Expression of the DGA1 transgene varied throughout the culture life cycle and evidence of growth dependent environmental silencing of the transgene was observed. This is to our knowledge the first example of silencing and subsequent resetting in a transgenic microalga. Results from this study add valuable insights into the efficacy of algal genetic engineering and use of these microorganisms as bio-platforms for chemical manufacture.

Synthetic regulation of eukaryotic gene expression by noncoding RNA

Synthetic biological systems promise to combine the spectacular diversity of biological functionality with engineering principles to design new life to address many pressing needs. As these engineered systems advance in sophistication, there is ever-greater need for customizable, situation-specific expression of desired genes. However, existing gene control platforms are generally not modular, or do not display performance requirements required for robust phenotypic responses to input signals. This work expands the capabilities of eukaryotic gene control in two important directions.

For development of greater modularity, we extend the use of synthetic self-cleaving ribozyme switches to detect changes in input protein levels and convey that information into programmed gene expression in eukaryotic cells. We demonstrate both up- and down-regulation of levels of an output transgene by more than 4-fold in response to rising input protein levels, with maximal output gene expression approaching the highest levels observed in yeast. In vitro experiments demonstrate protein-dependent ribozyme activity modulation. We further demonstrate the platform in mammalian cells. Our switch devices do not depend on special input protein activity, and can be tailored to respond to any input protein to which a suitable RNA aptamer can be developed. This platform can potentially be employed to regulate the expression of any transgene or any endogenous gene by 3’ UTR replacement, allowing for more complex cell state-specific reprogramming.

We also address an important concern with ribozyme switches, and riboswitch performance in general, their dynamic range. While riboswitches have generally allowed for versatile and modular regulation, so far their dynamic ranges of output gene modulation have been modest, generally at most 10-fold. We address this shortcoming by developing a modular genetic amplifier for near-digital control of eukaryotic gene expression. We combine ribozyme switch-mediated regulation of a synthetic TF with TF-mediated regulation of an output gene. The amplifier platform allows for as much as 20-fold regulation of output gene expression in response to input signal, with maximal expression approaching the highest levels observed in yeast, yet being tunable to intermediate and lower expression levels. EC50 values are more than 4 times lower than in previously best-performing non-amplifier ribozyme switches. The system design retains the modular-input architecture of the ribozyme switch platform, and the near-digital dynamic ranges of TF-based gene control.

Together, these developments suggest great potential for the wide applicability of these platforms for better-performing eukaryotic gene regulation, and more sophisticated, customizable reprogramming of cellular activity.

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

An Asp49 Phospholipase A2 From Snake Venom Induces Cyclooxygenase-2 Expression And Prostaglandin E2 Production Via Activation Of Nf-κb, P38mapk, And Pkc In Macrophages.

Phospholipases A2 (PLA2) are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PG)E2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2). Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

Diet Carotenoid Lutein Modulates The Expression Of Genes Related To Oxygen Transporters And Decreases Dna Damage And Oxidative Stress In Mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.

Identification Of Target Genes Using Gene Expression Profile Of Granulocytes From Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Differential gene expression analysis by suppression subtractive hybridization with correlation to the metabolic pathways involved in chronic myeloid leukemia (CML) may provide a new insight into the pathogenesis of CML. Among the overexpressed genes found in CML at diagnosis are SEPT5, RUNX1, MIER1, KPNA6 and FLT3, while PAN3, TOB1 and ITCH were decreased when compared to healthy volunteers. Some genes were identified and involved in CML for the first time, including TOB1, which showed a low expression in patients with CML during tyrosine kinase inhibitor treatment with no complete cytogenetic response. In agreement, reduced expression of TOB1 was also observed in resistant patients with CML compared to responsive patients. This might be related to the deregulation of apoptosis and the signaling pathway leading to resistance. Most of the identified genes were related to the regulation of nuclear factor κB (NF-κB), AKT, interferon and interleukin-4 (IL-4) in healthy cells. The results of this study combined with literature data show specific gene pathways that might be explored as markers to assess the evolution and prognosis of CML as well as identify new therapeutic targets.