Expression Of EGFP Reporter Gene Research Articles

Application of Hypoxia-Driven Dual-Reporter Model to Explore the Effect of Hypoxia on Radiosensitivity of Human Nasopharyngeal Carcinoma Cells

<h3>Purpose/Objective(s)</h3> hypoxia has been commonly observed in human nasopharyngeal carcinoma (NPC) which is associated with resistance to radiation and malignant phenotypes. The purpose of the study was to apply our build-in hypoxia-driven dual reporter model to explore the effect of hypoxia on radiosensitivity <b>in vitro</b> and <b>in vivo</b>. <h3>Materials/Methods</h3> The modalities to research the effect of hypoxia <b>in vitro</b> includes clonogenic survival assay and γH2AX foci formation. Tumor growth delay with radiation and/or cisplatin, imaging study on spatial biodistribution between blood perfusion (Hoechst 33342), endogenous (eGFP and CA9) and exogenous (pimonidazole) hypoxia markers were used to investigate the effect of hypoxia <b>in vivo</b>. <h3>Results</h3> The constructed hypoxia-driven dual-reporter model contains 9 repeats of hypoxia responsive element and fusion gene of 9xHRE-HSV1-TKeGFP in NPC cells (abbreviated as SUNE-1/HRE cells) in which hypoxia-induced molecular events can be visualized by fluorescence or nuclear imaging techniques. There is no difference in radiosensitivity between SUNE1/Parental and SUNE1/HRE cells, while cisplatin significantly increases radiosensitivity in both cell lines. γH2AX serves as a surrogate for unrepaired DSB. When the cells are exposed to 0.5% O₂, eGFP is observed only in SUNE1/HRE cells. DSB repair rate under hypoxia is similar in SUNE1/Parental and SUNE1/HRE cells. The number of γH2AX foci is dramatically higher in the presence of cisplatin compared to that given irradiation alone under the condition of normoxia or hypoxia. It indicates that the addition of cisplatin enhances radiosensitivity by compromising the ability of cells to repair DSB. Cisplatin alone shows modest whereas irradiation moderate effect on delayed tumor growth. Combined treatment of irradiation and cisplatin further enhances the effect on tumor growth delay. High expression of eGFP is observed in the nonperfused regions. The spatial biodistribution of hypoxia-induced expression of eGFP reporter gene is similar to other endogenous or exogenous hypoxia biomarkers, i.e., CA9 and pimonidazole. <h3>Conclusion</h3> The build-in hypoxia-driven dual reporter 9HRE-HSV1-TKeGFP hypoxia research model does not modify intrinsic radiosensitivity of SUNE1 cells under the condition of normoxia or hypoxia, suggesting it be used as preclinical model to explore the therapeutic efficacy of various treatment modalities to overcome hypoxia by fluorescence or nuclear imaging techniques.

A study on the use of strain-specific and homologous promoters for heterologous expression in industrial Saccharomyces cerevisiae strains

Polymorphism is well known in Saccharomyces cerevisiae strains used for different industrial applications, however little is known about its effects on promoter efficiency. In order to test this, five different promoters derived from an industrial and a laboratory (S288c) strain were used to drive the expression of eGFP reporter gene in both cells. The ADH1 promoter (PADH1) in particular, which showed more polymorphism among the promoters analyzed, also exhibited the highest differences in intracellular fluorescence production. This was further confirmed by Northern blot analysis. The same behavior was also observed when the gene coding for secreted α-amylase from Cryptococcus flavus was placed under the control of either PADH1. These results underline the importance of the careful choice of the source of the promoter to be used in industrial yeast strains for heterologous expression.

Combined sense-antisense Alu elements activate the EGFP reporter gene when stable transfection.

Alu elements in the human genome are present in more than one million copies, accounting for 10% of the genome. However, the biological functions of most Alu repeats are unknown. In this present study, we detected the effects of Alu elements on EGFP gene expression using a plasmid system to find the roles of Alu elements in human genome. We inserted 5'-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3' sequences into the pEGFP-C1 vector to construct expression vectors. We altered the copy number of Alus, the orientation of the Alus, and the presence of an enhancer (4TMI) in the inserted 5'-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3' sequences. These expression vectors were stably transfected into HeLa cells, and EGFP reporter gene expression was determined. Our results showed that combined sense-antisense Alu elements activated the EGFP reporter gene in the presence of enhancers and stable transfection. The combined sense-antisense Alu vectors carrying four copies of Alus downstream of inserted CMV induced much stronger EGFP gene expression than two copies. Alus downstream of inserted CMV were replaced to AluJBs (having 76% homology with Alu) to construct expression vectors. We found that combined sense-antisense Alu (or antisense AluJB) vectors induced strong EGFP gene expression after stable transfection and heat shock. To further explore combined sense-antisense Alus activating EGFP gene expression, we constructed Tet-on system vectors, mini-C1-Alu-sense-sense and mini-C1-Alu-sense-antisense (EGFP gene was driven by mini-CMV). We found that combined sense-antisense Alus activated EGFP gene in the presence of reverse tetracycline repressor (rTetR) and doxycycline (Dox). Clone experiments showed that Mini-C1-Alu-sense-antisense vector had more positive cells than that of Mini-C1-Alu-sense-sense vector. The results in this paper proved that Alu repetitive sequences inhibited gene expression and combined sense-antisense Alus activated EGFP reporter gene when Alu transcribes, which suggests that Alus play roles in maintaining gene expression (silencing genes or activating genes) in human genome.

Functional and Comparative Genomics of Hoxa2 Gene cis-Regulatory Elements: Evidence for Evolutionary Modification of Ancestral Core Element Activity.

Hoxa2 is an evolutionarily conserved developmental regulatory gene that functions to specify rhombomere (r) and pharyngeal arch (PA) identities throughout the Osteichthyes. Japanese medaka (Oryzias latipes) hoxa2a, like orthologous Hoxa2 genes from other osteichthyans, is expressed during embryogenesis in r2–7 and PA2-7, whereas the paralogous medaka pseudogene, ψhoxa2b, is expressed in noncanonical Hoxa2 domains, including the pectoral fin buds. To understand the evolution of cis-regulatory element (CRE) control of gene expression, we conducted eGFP reporter gene expression studies with extensive functional mapping of several conserved CREs upstream of medaka hoxa2a and ψhoxa2b in transient and stable-line transgenic medaka embryos. The CREs tested were previously shown to contribute to directing mouse Hoxa2 gene expression in r3, r5, and PA2-4. Our results reveal the presence of sequence elements embedded in the medaka hoxa2a and ψhoxa2b upstream enhancer regions (UERs) that mediate expression in r4 and the PAs (hoxa2a r4/CNCC element) or in r3–7 and the PAs ψhoxa2b r3–7/CNCC element), respectively. Further, these elements were shown to be highly conserved among osteichthyans, which suggests that the r4 specifying element embedded in the UER of Hoxa2 is a deeply rooted rhombomere specifying element and the activity of this element has been modified by the evolution of flanking sequences that redirect its activity to alternative developmental compartments.

Modification of a salmonid alphavirus replicon vector for enhanced expression of heterologous antigens.

A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5' end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 3' end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future.

Oct4‐EGFP reporter gene expression marks the stem cells in embryonic development and in adult gonads of transgenic medaka

Maintenance of pluripotency in stem cells is tightly regulated among vertebrates. One of the key genes in this process is oct4, also referred to as pou5f1 in mammals and pou2 in teleosts. Pou5f1 evolved by duplication of pou2 early in the tetrapod lineage, but only monotremes and marsupials retained both genes. Either pou2 or pou5f1 was lost from the genomes of the other tetrapods that have been analyzed to date. Consequently, these two homologous genes are often designated oct4 in functional studies. In most vertebrates oct4 is expressed in pluripotent cells of the early embryo until the blastula stage, and later persist in germline stem cells until adulthood. The isolation and analysis of stem cells from embryo or adult individuals is hampered by the need for reliable markers that can identify and define the cell populations. Here, we report the faithful expression of EGFP under the control of endogenous pou2/oct4 promoters in transgenic medaka (Oryzias latipes). In vivo imaging in oct4-EGFP transgenic medaka reveals the temporal and spatial expression of pou2 in embryos and adults alike. We describe the temporal and spatial patterns of endogenous pou2 and oct4-EGFP expression in medaka with respect to germline and adult stem cells, and discuss applications of oct4-EGFP transgenic medaka in reproductive and stem cell biology.

A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector

siRNA technique has been widely used to study the gene functions and to develop disease therapeutics. One of the challenges of RNAi application is to obtain the most effective target specific siRNA sequences. Currently the process to select and validate optimal siRNA sites for a given gene, which is usually based on screening by using Western blot, Northern blot or Q-PCR, remains empirical and time consuming. Although few fluorescence-based siRNA sequence selection systems have proven useful, the rapid and efficient screening of siRNA target sites is still challenging. In the paper, we developed a quick and efficient method to screen siRNA target sites with a novel single vector system, which contains the following cassettes: (1) an eGFP reporter gene expression cassette followed by a multiple cloning site and SV40 pA for insertion of a target sequence; (2) siRNA expression cassette containing a dual PoI III promoter driving in opposite directions; and (3) an internal loading control, mCherry reporter gene. Based on this one-step transfection with single vector system, we could rapidly screen effective, target specific siRNA fragments in an unbiased manner by judging the fluorescence intensity ratio of eGFP to mCherry. The generation of this novel vector system will promote the application of siRNA in basic research and disease therapy.

Ultrasound microbubbles enhancing the transfection of recombinant adeno-ussociated virus vector to retinal ganglion cells in vivo

Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adeno-associated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo. Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A, B, C, D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 μl phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 μl recombinant adeno-associated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5μl rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects. Results Green fluorescence can be observed exactly in labeled RGC in B,C, and D groups. The AOD and transfection rate in group D was (95.02 ± 7. 25)% and (20. 10±0. 74)% , respectively; which were higher than those in group B and C (F=25. 970,25. 799;P 0. 05). Conclusion Under the condition of low frequency and with certain energy, ultrasound-mediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats. Key words: Contrast Media/therapeutic use; Retinal ganglion cells; Gene transfer techniques; Green fluorescent proteins/agonists; Animal experimentation

Tet system in the brain: Transgenic rats and lentiviral vectors approach

Local and regulated expression of exogenous genes in the central nervous system is one of the major challenges of modern neuroscience. We have approached this issue by applying the inducible tetracycline system to regulate the expression of EGFP reporter gene in double transgenic rats. We have obtained a strong induction of EGFP only in male testes, which correlated with a high level of rtTA expression only in this organ. To overcome the problem of lack of rtTA protein in the transgenic rat brain, we have delivered this Tet system activator with lentiviral vectors into the dentate gyrus of hippocampus of transgenic EGFP rats. As a result, after systemic application of doxycycline we have obtained inducible, stable and restricted to the desired brain region expression of EGFP. An advantage of this strategy is that the transgene is located in the same genetic milieu in every cell of the transgenic organism. This is crucial to obtain uniform expression of the regulated gene within the target brain structure. Combination of rat transgenesis and lentiviral vectors is a novel approach enabling precise spatiotemporal regulation of genes of interest strictly in the brain structure of choice or in other tissues.

300. Gene Therapy for Gastric Cancer Cells Via CEA-Targeted Adv-FZ33

IntroductionAt present, adenoviral vectors have been widely used for gene transfer into tumor cells primarily because they can be produced in high titers and can infect many different cell types. However, a major problem when utilizing the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency due to weak expression of the adenovirus receptor, coxsackie– adenovirus receptor (CAR) in cancer cells, or side effects due to expression of CAR in normal cells. There is an urgent requirement to improve the specificity of gene delivery via adenoviral vectors in the context of cancer gene therapy.Experimental designWe constructed a fiber modified Ad5 vector (Adv-FZ33) in which a IgG-binding domain (Z33) derived from staphylococcal protein A was inserted into the HI loop of knob protein. We then evaluated on both in vitro and in vivo levels the extent of retargeting towards and therapeutic effectiveness against CEA-positive gastric cancers when using the fully human CEA antibody (C2-45) complex with this modified vector.ResultsIn vitro LacZ or EGFP reporter gene expression after infection with Adv-FZ33 vectors conjugated to anti-CEA mAb was approximately 20 times higher than that obtained with vector conjugated to the control mAb (human IgG4). We generated Ax3CAUP-FZ33, which is an Adv-FZ33 derivative vector expressing a therapeutic gene, namely E. coli uracil phosphoribosyltransferase (UPRT) which converts 5FU directly to 5-fluorouridine monophosphate. When conjugated with the anti-CEA mAb, C2-45, Ax3CAUP-FZ33 enhanced the cytotoxicity of 5FU (19.7-fold in terms of IC50 values) against gastric cancer cells. In a nude mouse peritoneal dissemination model, use of Ax3CAUP- FZ33 conjugated with anti-CEA mAb C2-45, in combination with 5FU administration, resulted in greatly enhanced anti-tumor effects against CEA-positive cancer cells. Tumor growth in mice treated with Ax3UP-FZ33/C2-45/5FU was significantly suppressed, and tumor volumes were less than a quarter of those of the control IgG4 group (total weight, 0.12 ± 0.11 g; P < 0.05). Furthermore, we examined the survival effects of Ax3UP-FZ33 conjugated with anti- CEA mAb plus 5FU in mice with peritoneal disseminated gastric cancer. The median survival time of the Ax3CAUP-FZ33/C2-45/5FU group was significantly longer than those of the PBS and 5FU only treatment groups (p<0.01, versus PBS and 5FU only groups). On the other hand, no significant difference was present between the Ax3CAUP-FZ33/5FU or Ax3CAUP-FZ33/IgG4/5FU groups and the PBS or 5FU only groups.ConclusionsThese data suggest that CEA-targeted FZ33-mutant adenovirus- mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers. This novel strategy may be a promising tool for cancer gene therapy.

Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing

The genetic elements that are responsible for establishing a transcriptionally competent, open chromatin structure at a region of the genome that consists only of ubiquitously expressed, housekeeping genes are currently unknown. We demonstrate for the first time through functional analysis in stably transfected tissue culture cells that transgenes containing methylation-free CpG islands spanning the dual divergently transcribed promoters from the human TATA binding protein ( TBP)–proteasome component-B1 ( PSMB1) and heterogeneous nuclear ribonucleoprotein A2/B1 ( HNRPA2B1)–heterochromatin protein 1Hs-γ (chromobox homolog 3, CBX3) gene loci are sufficient to prevent transcriptional silencing and a variegated expression pattern when integrated within centromeric heterochromatin. In addition, only transgene constructs extending over both the HNRPA2B1 and the CBX3 promoters, and not the HNRPA2B1 promoter alone, were able to confer high and stable long-term EGFP reporter gene expression. These observations suggest that methylation-free CpG islands associated with dual, divergently transcribed promoters possess an independent dominant chromatin opening function and may therefore be major determinants in establishing and maintaining a region of open chromatin at housekeeping gene loci.