Enhanced Green Fluorescent Protein Gene Expression Research Articles

Enhancement of periodontal tissue regeneration by transplantation of osteoprotegerin-engineered periodontal ligament stem cells.

IntroductionThe objective of the present study was to evaluate the capacity of a tissue-engineered complex of human osteoprotegerin (hOPG)-transfected periodontal ligament stem cells (PDLSCs) seeding on beta-tricalcium phosphate (β-TCP) to regenerate alveolar bone defects in New Zealand rabbits.MethodsPDLSCs were isolated from rabbit periodontal ligament tissues and expanded in vitro to enrich PDLSC numbers, and their proliferative activities and differentiation capability were evaluated under specific induction conditions. Lentiviral vector containing hOPG and enhanced green fluorescent protein (EGFP) was constructed by using Gateway technology and transfected into rabbit PDLSCs. The expression of hOPG was determined with quantitative real-time reverse transcription-polymerase chain reaction and Western blot. The PDLSCs with or without engineered hOPG were seeded on β-TCP scaffolds prior to transplantation. Morphological characterization of cells and materials was done by scanning electron microscope. Twenty rabbits with alveolar bone defects were randomly allocated into four groups and transplanted with β-TCP, PDLSCs/β-TCP, and hOPG-transfected PDLSCs/β-TCP or were left untreated as a control. Animals were sacrificed 12 weeks after operation for histological observation and histomorphometric analysis.ResultsPDLSCs expressed STRO-1 and vementin and favored osteogenesis and adipogenesis in conditioned media. Expressions of hOPG were significantly upregulated after transfection of the lentiviral vector into PDLSCs. PDLSCs attached and spread well on β-TCP, and there was no significant difference in growth of PDLSCs on β-TCP between the hOPG transfection group and the non-transfection group. The histological observation and histomorphometric analysis showed that the hOPG-transfected PDLSCs/β-TCP complex exhibited an earlier mineralization and more bone formation inside the scaffold than control, β-TCP, and PDLSCs/β-TCP complexes. Implantation of hOPG-transfected PDLSCs contributed to new bone formation as determined by EGFP gene expression under circularly polarized light microscopy.ConclusionsThe present study demonstrated the feasibility of β-TCP scaffolds for primary PDLSC culture and expression of hOPG gene in vitro and in vivo, and hOPG-transfected PDLSCs could serve as a potential cell source for periodontal bone regeneration, which may shed light on the potential of systemic hOPG gene therapy in combination with PDLSC tissue engineering as a good candidate in periodontal tissue engineering for alveolar bone regeneration.

Cloning and Activity Analysis of Promoter of RanGTPase Gene from Shrimp, Marsupenaeus japonicus

RanGTPases, one superfamily of small G protein family, have been demonstrated to be involved in the innate immune system and they are up-regulated after pathogen infection. However, the regulation mechanism of RanGTPases remains unclear. In this study, the 5′ flanking region of shrimp RanGTPase gene was cloned and sequenced. To detect the activity of the promoter, pIZΔIE/EGFP was modified from pIZ/V5-His by replacing the OPIE2 promoter with this promoter and inserting enhanced green fluorescence protein (EGFP) gene downstream. The promoter can drive the expression of reporter EGFP gene in Hi5 insect cells, indicating the promoter has activity. These results extend our previous findings and provide insights into the molecular regulation of RanGTPase gene expression, which will be helpful for shrimp viral disease control.

Enhancing DNA delivery into the skin with a motorized microneedle device

The purpose of this study was to evaluate a motorized microneedle device in delivery of DNA into skin for gene expression. A plasmid DNA encoding both luciferase (Luc) and enhanced green fluorescent protein (EGFP) was delivered into rat skin by puncturing the skin with the microneedle device. Puncturing rat skin with a pre-applied DNA solution on the skin showed much higher luciferase gene expression than that with the procedure of puncturing the skin first then applied the DNA solution. The microneedle puncturing method was more efficient than intradermal injection method in generating high gene expression in the skin. There was no significant difference in the skin gene expression when rat skin was punctured with the microneedle device of different microneedle lengths (0.25mm, 0.5mm or 0.75mm). On the other hand, there was a significant difference in the skin gene expression between the short (10s) and the long puncturing durations (30 or 60s), with longer puncturing duration showed higher gene expression. Puncturing the skin with longer needles (0.75mm) caused some skin damage, while puncturing the skin with shorter microneedle length (0.25mm) caused only minimal skin damage. The EGFP gene expression was observed predominately in the epidermis layer of the skin from the puncturing method in delivery of DNA into the skin. In summary, the motorized microneedle device could have great potential in skin gene delivery.

Screening of promoters from rhizosphere metagenomic DNA using a promoter‐trap vector and flow cytometric cell sorting

We constructed a facilitative and efficient promoter-trap vector, pCM-EGFP, for capturing and analyzing functional promoters from environmental DNA. The pCM-EGFP vector showed good chloramphenicol sensitivity and no enhanced green fluorescent protein (EGFP) gene expression. Promoter libraries were constructed for screening promoters responding to naringenin, a key molecule released from plant roots. After electroporation, E. coli transformants were incubated in LB broth containing chloramphenicol (10 μg/ml) to select against transformants with no cloned promoter. E. coli cells were sorted using flow cytometry without naringenin, and then sorted again with high fluorescence after incubation in LB broth with naringenin (1 mM) at 28 °C for 12 h. The inducible properties of approximately 400 sorted cells were evaluated, with most cells showing only strong EGFP gene expression without inducible properties. Two clones (5-4E and 15-3D) displayed naringenin inducibility, and both contained a promoter bounded by a TetR-family regulator. The regulator knock-out mutant of the 5-4E clone lost its ability to be induced by naringenin. In conclusion, the pCM-EGFP vector may be used as an efficient promoter-trap vector and a combination of the vector with flow cytometric cell sorting was demonstrated to be an useful method for screening promoters responding to specific conditions or inducers.

Site-specific gene transfer into the rat spinal cord by photomechanical waves

Nonviral, site-specific gene delivery to deep tissue is required for gene therapy of a spinal cord injury. However, an efficient method satisfying these requirements has not been established. This study demonstrates efficient and targeted gene transfer into the spinal cord by using photomechanical waves (PMWs), which were generated by irradiating a black laser absorbing rubber with 532-nm nanosecond Nd:YAG laser pulses. After a solution of plasmid DNA coding for enhanced green fluorescent protein (EGFP) or luciferase was intraparenchymally injected into the spinal cord, PMWs were applied to the target site. In the PMW application group, we observed significant EGFP gene expression in the white matter and remarkably high luciferase activity only in the spinal cord segment exposed to the PMWs. We also assessed hind limb movements 24 h after the application of PMWs based on the Basso-Beattie-Bresnahan (BBB) score to evaluate the noninvasiveness of this method. Locomotor evaluation showed no significant decrease in BBB score under optimum laser irradiation conditions. These findings demonstrated that exogenous genes can be efficiently and site-selectively delivered into the spinal cord by applying PMWs without significant locomotive damage.

Fusion of EGFP and porcine α 1,3GT genes decrease GFP expression

Objective To investigate the effect of fusion proteins expressed by the fused gene of porcine α 1, 3 galactosyltransferase (α1, 3 GT) and enhanced green fluorescent protein (EGFP) on the green fluorescence intensity of EGFP. Methods The fragment containing α 1, 3GT was firstly recovered after the pcDNA3.1- α 1, 3GT recombinant vector were digested with Bam HI and Eco RI, and then, the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes. The new recombinant eukaryotic expression pEGFP/α 1, 3GT vector was obtained and sequenced. The pEGFP/α 1, 3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT, and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry. Results The positive percentage of A549 was 80.5%, and that of 293 FT was 86.5% 48 hours after the two cell lines both were transfected by pEGFP-N1. The positive percentage of A549 was 75.8%, and that of 293 FT was 81.2% 48 hours after the two cell lines were transfected by pEGFP/α 1, 3GT. The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 1.21 and 0.956, respectively when compared with that of A549 without transfection. Meanwhile, the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 7.66 and 1.00, respectively when compared with that of 293FT cells without transfection. Conclusions These results suggested that the expression of EGFP gene fused with porcine α 1, 3GT gene was partly inhibited.

Layer-by-layer assembly of small interfering RNA and poly(ethyleneimine) for substrate-mediated electroporation with high efficiency

Electroporation microarrays have been developed for the high-throughput transfection of expression constructs and small interfering RNAs (siRNAs) into living mammalian cells. These techniques have potential to provide a platform for the cell-based analysis of gene functions. One of the key issues associated with microarray technology is the efficiency of transfection. The capability of attaining reasonably high transfection efficiency is the basis for obtaining functional data without false negatives. In this study, we aimed at improving the transfection efficiency in the system that siRNA loaded on an electrode is electroporated into cells cultured directly on the electrode. The strategy we adopted here is to increase the surface density of siRNA loaded onto electrodes. For this purpose, the layer-by-layer assembly of siRNA and cationic polymers, branched or linear form of poly(ethyleneimine), was performed. The multilayer thus obtained was characterized by infrared reflection-adsorption spectroscopy and surface plasmon resonance analysis. Transfection efficiency was evaluated in a system that siRNA specific for enhanced green fluorescent protein (EGFP) was electroporated on the electrode into human embryonic kidney cells stably transformed with the EGFP gene. The suppression of EGFP expression was assessed by fluorescence microscopy and flow cytometry. Our data showed that the layer-by-layer assembly of siRNA with branched poly(ethyleneimine) facilitated to increase the surface density of loaded siRNA. As a result, the expression of EGFP gene in the electroporated cells was suppressed much more on the electrodes with the multilayer of siRNA than that with the monolayer.

Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo

This study was conducted to investigate the efficacy and safety of ultrasound (US)-targeted microbubble (MB) destruction (UTMD)-mediated rAAV2-CMV-EGFP transfection to cultured human retinal pigment epithelium (RPE) cells in vitro and to the rat retina in vivo. In the in vitro study, cultured human RPE cells were exposed to US under different conditions with or without MBs. Furthermore, the effect of UTMD on rAAV2-CMV-EGFP itself and on cells was evaluated. In the in vivo study, gene transfer was examined by injecting rAAV2-CMV-EGFP into the subretinal space of rats with or without MBs and then exposed to US. We investigated enhanced green fluorescent protein (EGFP) expression in vivo by stereomicroscopy and performed quantitative analysis using Axiovision 3.1 software. Hematoxylin and eosin staining and frozen sections were used to observe tissue damage and location of the EGFP gene expression. In the in vitro study, the transfection efficiency of rAAV2-CMV-EGFP under optimal UTMD was significantly higher than that of the control group (P=0.000). Furthermore, there was almost no cytotoxicity to the cells and to rAAV2-CMV-EGFP itself. In the in vivo study, UTMD could be used safely to enhance and accelerate the transgene expression of the retina. Fluorescence expression was mainly located in the retinal layer. UTMD is a promising method for gene delivery to the retina.

312 CONSTRUCTION OF SPECIFICALLY EXPRESSED VECTOR IN MAMMARY GLAND FOR lacS AND ITS TRANSFECTION INTO BOVINE FETAL FIBROBLASTS MEDIATED BY LIPOSOME

This study was designed to optimize conditions for transfection of a mammary gland specific transgene into bovine fetal fibroblasts. Transfection of Sulfolobus solfataricus β-glycosidase gene (lacS) was mediated by liposome. Neomycin resistance (Neor) and enhanced green fluorescent protein (EGFP) gene were used as genetic markers to screen transgenic somatic cells. A 0.92-kb fragment of bovine β-lactoglobulin gene sequence was obtained from bovine genome by PCR amplification and was inserted into the T site of pMD19-Simple T plasmid. 1.49 kb of lacS gene coding sequence was cloned from Sulfolobus solfataricus genome by PCR amplification and inserted into the pUC19 plasmid. The coding sequence of Neor was derived by PCR amplification from pIRES2-EGFP plasmid and inserted into the BamHI/NheI site of pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a Neor and an EGFP gene, which were linked by an internal ribosome entry site sequence downstream of the cytomegalovirus (CMV) promoter. Finally, the vector pNIE was assembled into the pUC19 plasmid, thus creating a pBLI vector, which contained the Neor and EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the lacS gene regulated by bovine β-lactoglobulin promoter for specific expression in mammary gland. Bovine fetal fibroblasts (bFF) were isolated from the ear skin of female fetuses at the age of 2 to 3 months. The cells proliferated well and grew normally in culture, with typical fibroblast morphology and growth curve. The effects of different concentrations of transfection and pBLI were compared on the efficiency of transfection. The passage 4 bFF cells at 70 to 80% confluency were transfected in a 24-well culture plate. 2 × 105 cells were cultured in DMEM with 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 μg of pBLI using transfection (1, 2, 3, 4, 5, and 6 μL) for 48 h, respectively. The transfected cells were cultured for 48 h before adding G418 at concentrations of 200, 300, 400, 500, 600, 700, 800, and 900 μg mL–1 for 14 d, respectively. Positive cell colonies were selected and purified through both the expression of Neor and EGFP gene under a fluorescence microscopy. The selected colonies were propagated in DMEM containing 300 μg mL–1 G418. The results showed that bright green fluorescence could be detected at 48 h after transfection. 1.0 μg of pBLI plasmid and 3 μL of transfection yielded the desirable efficiency of transfection. More transgenic bFF colonies were selected by G418 at the concentration of 800 μg mL–1. In conclusion, a specifically expressed vector in mammary gland for lacS gene was successfully constructed, transfection parameters were developed, and efficient screening measures were established for detecting transgenic somatic cells.

Stable EGFP gene expression in C6 glioma cell line after transduction with HIV-1-based lentiviral vector

To establish a stable C6/EGFP glioma cell line for studies on glioma. The C6 glioma cell line was transfected with the human immunodeficiency virus type I (HIV-1) based lentivirus vector containing two enhancer-promoters CMV and EF1α. Enhanced green fluorescent protein (EGFP)-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by Polymerase chain reaction (PCR) and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. A C6/EGFP cell line expressing EGFP as a marker is established, in which the EGFP gene is integrated into the genome. This cell line can be served as a promising tool for further basic research and gene therapy studies.

Localization of green fluorescent protein in mouse preimplantation embryos

The localization of enhanced green fluorescent protein (EGFP) was studied in preimplantation embryos obtained from reciprocal mating of hemizygous C57Bl/6-Tgn (ACTbEGFP)1Osb/J mice with C57Bl/6 mice. Specific fluorescence of EGFP was observed in all oocytes and embryos obtained from transgenic females during all preimplantation stages and in embryos inheriting the EGFP gene from transgenic males starting from the 8 blastomere stage during the compactization period. EGFP mRNA or EGFP synthesized during oogenesis can be retained in embryos during the entire preimplantation period, while expression of EGFP gene transferred from the father coincides with the onset of compactization. The possibility of using these embryos in experimental mammalian embryology is discussed.

Functional analysis of the Myostatin gene promoter in sheep

Compared with the understanding for the functional mechanism of the myostatin gene, little is known about the regulatory mechanism of the myostatin gene transcription and expression. To better understand the function of the myostatin gene promoter (MSTNpro) in the transcriptional regulation of the myostatin gene and to further investigate the transcriptional regulation mechanism of the myostatin gene, the promoter region of the myostatin gene in sheep has been cloned in our recent study (AY918121). In this study, the wild (W) type MSTNPro(W)-EGFP vectors and E-box (E) (CANNTG) mutant (M) type MSTNPro(E(3+5+7)M)-EGFP vectors were constructed and the transcriptional regulation activities were compared by detecting the fluorescent strength of EGFP (enhanced green fluorescent protein) in C2C12 myoblasts (or myotubes) and sheep fibroblasts transfected with the vectors. Results showed that the 0.3-1.2 kb sheep myostatin promoter could activate the transcription and expression of EGFP gene in C2C12 myoblasts to different extent and the 1.2 kb promoter was the strongest. However, fluorescence was not observed in the sheep fibroblasts transfected with the 1.2 kb sheep myostatin promoter. These results suggested that the specific nature of the myostatin gene expression in skeletal muscle was attributed to the specific nature of the myostatin promoter activity. The increasing growth density of C2C12 myoblasts inhibited the transcriptional regulation activity of the wild type sheep myostatin promoter by a mechanism of feedback. The transcriptional regulation activity of the 1.2 kb wild type sheep myostatin promoter increased significantly after C2C12 myoblasts were differentiated, while the activity of 1.2 kb E(3+5+7)-mutant type myostatin promoter had no obvious change. This result suggested that MyoD may be responsible for the difference of the myostatin gene transcription and expression between growing and differentiating conditions by binding to E-box of the myostatin promoter.

Tumor-specific Activation of Human Telomerase Reverses Transcriptase Promoter Activity by Activating Enhancer-binding Protein-2β in Human Lung Cancer Cells

The up-regulated expression and telomerase activity of human telomerase reverse transcriptase (hTERT) are hallmarks of tumorigenesis. The hTERT promoter has been shown to promote hTERT gene expression selectively in tumor cells but not in normal cells. However, little is known about how tumor cells differentially activate hTERT transcription and induce telomerase activity. In this study, we identified activating enhancer-binding protein-2beta (AP-2beta) as a novel transcription factor that specifically binds to and activates the hTERT promoter in human lung cancer cells. AP-2beta was detected in hTERT promoter DNA-protein complexes formed in nuclear extracts prepared only from lung cancer cells but not from normal cells. We verified the tumor-specific binding activity of AP-2beta for the hTERT promoter in vitro and in vivo and detected high expression levels of AP-2beta in lung cancer cells. We found that ectopic expression of AP-2beta reactivated hTERT promoter-driven reporter green fluorescent protein (GFP) gene and endogenous hTERT gene expression in normal cells, enhanced GFP gene expression in lung cancer cells, and prolonged the life span of primary lung bronchial epithelial cells. Furthermore, we found that inhibition of endogenous AP-2beta expression by AP-2beta gene-specific small interfering RNAs effectively attenuated hTERT promoter-driven GFP expression, suppressed telomerase activity, accelerated telomere shortening, and inhibited tumor cell growth by induction of apoptosis in lung cancer cells. Our results demonstrate the tumor-specific activation of the hTERT promoter by AP-2beta and imply the potential of AP-2beta as a novel tumor marker or a cancer therapeutic target.

In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery

RNA interference (RNAi)-mediated gene silencing approaches appear very promising for therapies based on the targeted inhibition of disease-relevant genes. The major hurdle to the therapeutic development of RNAi strategies remains, however, the efficient delivery of the RNAi-inducing molecules, the short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to the target tissue. With respect to cancer treatment the development of efficient delivery methods into solid tumors appears as a critical issue. However, very few studies have addressed this problem. In this study we have investigated the contribution of electrically mediated delivery of siRNA into murine tumors stably expressing an enhanced green fluorescent protein (EGFP) target reporter gene. The silencing of EGFP gene expression was quantified over time by fluorescence imaging in the living animal. Our study indicates that electric field can be used as an efficient method for siRNA delivery and associated gene silencing into cells of solid tumors in vivo.

Inhibition of RAW264.7 macrophage inflammatory cytokines release by small haparin RNAi targeting TLR4

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-alpha) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37+/-8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-alpha released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.

Tetracycline‐inducible gene expression in nuclear transfer embryos derived from porcine fetal fibroblasts transformed with retrovirus vectors

A critical problem of transgenic livestock production is uncontrollable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in transgenic animals. One of the best solutions for this problem may be use of controllable gene expression system. In this study, using retrovirus vectors designed to express the enhanced green fluorescent protein (EGFP) gene under the control of the tetracycline-inducible promoter, we examined whether the expression of the transgene could be controllable in fibroblast cells and nuclear transfer (NT) embryos of porcine. Transformed fibroblast cells were cultured in medium supplemented with or without doxycycline (a tetracycline analog) for 48 hr, and the induction efficiency was measured by comparing EGFP gene expression using epifluorescence microscopy and Western and Northern blot analyses. After the addition of doxycycline, EGFP expression increased up to 17-fold. The nuclei of transformed fibroblast cells were transferred into enucleated oocytes. Fluorescence emission data revealed strong EGFP gene expression in embryos cultured with doxycycline, but little or no expression in the absence of the antibiotic. Our results demonstrate the successful regulation of transgene expression in porcine nuclear transfer embryos, and support the application of an inducible expression system in transgenic pig production to solve the inherent problems of side-effects due to constitutive expression of the transgene.

Inhibition of rat RAW264.7 macrophage inflammatory cytokines release by small hairpin RNAi targeting Toll-like receptor

To construct a eukaryotic expression vector carrying the small hairpin RNA (shRNA) for Toll-like receptor 4 (TLR4) mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by rat RAW264.7 macrophages induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism. The H1 promotor and double BbsIrestrict endoenzyme site from the plasmid psiRNA-hH1neo were cloned into the reporter gene plasmid pEGFP-C1 at the MluIrestrict endoenzymic site, thus forming the plasmid pEGFP-H1/siRNA containing Bbs site and reporter EGFP gene. Then an oligo nuclear hairpin sequence targeting TLR4 gene was designed by the internet tool siRNA Wizard and then inserted into the plasmid pEGFP-H1/siRNA so as to form the plasmid pEGFP-H1/TLR4-siRNA. Rat macrophages of the line RAW264.7 were cultured and transfected with pEGFP-H1/TLR4-siRNA mediated by lipofectamine 2000. Another RAW264.7 cells were transfected with pEGFP-H1/control sequence-siRNA or blank plasmid. Lipopolysaccharide was added into the 3 kinds of culture fluid for 2 and 68 hours respectively. ELSA was used to detect the levels of tumor necrosis factor-alpha (TNF-alpha) in the supernatants. Restriction endonuclease analysis showed that the construction pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene was successful. The expression of EGFP gene was 50% +/- 8%. The TNF-alpha level of the TLR4-siRNA transfection group 2 hours and after transfection was 825 pg/ml +/- 136 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (2190 pg/ml +/- 359 pg/ml and 1265 pg/ml +/- 283 pg/ml respectively, both P < 0.01). The TNF-alpha level of the TLR4-siRNA transfection group 8 hours and after transfection was 1179 pg/ml +/- 240 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (4720 pg/ml +/- 227 pg/ml and 4689 pg/ml +/- 310 pg/ml respectively, both P < 0.01). shRNA targeting TLR4 gene can inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.

WPRE-mediated enhancement of gene expression is promoter and cell line specific

The success of gene therapy approaches relies on sufficiently high levels of expression of the therapeutic gene. However, if tissue specific or tumour specific gene expression is desired, a lower level of transgene expression usually has to be accepted due to the weakness of the majority of available tissue or tumour specific promoters. This obstacle can in part be overcome by the insertion of viral cis-acting elements that enhance gene expression in various expression vector contexts regardless of the respective promoter. We designed a series of murine leukaemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors that contain the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and evaluated its use by measuring enhanced green fluorescent protein (EGFP) levels and viral titres. In viral vector packaging cells, when the EGFP encoding gene was transcribed from the MLV promoter, incorporation of the WPRE resulted in a marked improvement of the vectors in terms of EGFP expression and virus titres. However, in infected cells after promoter conversion had taken place, the effect of the WPRE became promoter and cell line dependent. When the EGFP gene was transcribed from the heterologous mouse mammary tumour virus (MMTV) promoter the same beneficial role of the WPRE on transgene expression was observed in all eight cell lines tested. In contrast, when EGFP gene expression was driven by the murine whey acidic protein (WAP) promoter, the positive effect of the WPRE could only be observed in two cell lines whereas expression was actually reduced in the six other cell lines tested. This decrease of EGFP expression was not only demonstrated at the protein level but also manifested on the RNA level.

The flavonoid luteolin prevents lipopolysaccharide‐induced NF‐κB signalling and gene expression by blocking IκB kinase activity in intestinal epithelial cells and bone‐marrow derived dendritic cells

The nuclear factor (NF)-kappaB transcriptional system is a major effector pathway involved in inflammation and innate immune responses. The flavonoid luteolin is found in various herbal extracts and has shown anti-inflammatory properties. However, the mechanism of action and impact of luteolin on innate immunity is still unknown. We report that luteolin significantly blocks lipopolysaccharide (LPS)-induced IkappaB phosphorylation/degradation, NF-kappaB transcriptional activity and intercellular adhesion molecule-1 (ICAM-1) gene expression in rat IEC-18 cells. Using chromatin immunoprecipitation, we demonstrate that LPS-induced RelA recruitment to the ICAM-1 gene promoter is significantly reduced in luteolin-treated cells. Moreover, in vitro kinase assays show that luteolin directly inhibits LPS-induced IkappaB kinase (IKK) activity in IEC-18 cells. Using bone-marrow derived dendritic cells (BMDCs) isolated from interleukin (IL)-10(-/-) mice or from recently engineered transgenic mice expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-kappaB cis-elements (cis-NF-kappaB(EGFP)), we found that luteolin blocks LPS-induced IkappaB phosphorylation and IKK activity, and decreases EGFP, IL-12 and tumour necrosis factor-alpha gene expression. Moreover, intraperitoneal administration of luteolin significantly inhibited LPS-induced EGFP expression in both peripheral blood mononuclear cells and splenocytes isolated from cis-NF-kappaB(EGFP) mice. These results indicate that luteolin blocks LPS-induced NF-kappaB signalling and proinflammatory gene expression in intestinal epithelial cells and dendritic cells. Modulation of innate immunity by natural plant products may represent an attractive strategy to prevent intestinal inflammation associated with dysregulated innate immune responses.

Expression of the estrogen-inducible EGFP gene in aromatase-null mice reveals differential tissue responses to estrogenic compounds

Aromatase is an enzyme responsible for the conversion of androgen to estrogen. We genetically engineered an aromatase-deficient mouse (Ar −/− mouse) to express an enhanced green fluorescent protein (EGFP) gene in the uterus, ovary, adrenal and pituitary glands in a 17β-estradiol (E2)-inducible manner. In this study, we analyzed estrogenic activities of diethylstilbestrol, genistein, daidzein and E2 in the Ar −/− tissues by using the EGFP expression as an indicator. These analyses manifest differential responses of the tissues to the compounds and also allow to determine the relative estrogenic potency of the compounds to that of E2 in vivo. Furthermore, analyses of the EGFP expression in ERα-deficient mice suggested that the expression is ERα-dependent in the uterus and pituitary gland. In conclusion, the Ar −/− mouse carrying the E2-inducible EGFP gene is a valuable tool for quantitative analyses of natural and synthetic estrogenic compounds in vivo.