Green Fluorescent Protein Assay Research Articles

Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters-such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride-were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP.

Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cell-free protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.

High-throughput developability assays enable library-scale identification of producible protein scaffold variants

Proteins require high developability-quantified by expression, solubility, and stability-for robust utility as therapeutics, diagnostics, and in other biotechnological applications. Measuring traditional developability metrics is low throughput in nature, often slowing the developmental pipeline. We evaluated the ability of 10 variations of three high-throughput developability assays to predict the bacterial recombinant expression of paratope variants of the protein scaffold Gp2. Enabled by a phenotype/genotype linkage, assay performance for 105 variants was calculated via deep sequencing of populations sorted by proxied developability. We identified the most informative assay combination via cross-validation accuracy and correlation feature selection and demonstrated the ability of machine learning models to exploit nonlinear mutual information to increase the assays' predictive utility. We trained a random forest model that predicts expression from assay performance that is 35% closer to the experimental variance and trains 80% more efficiently than a model predicting from sequence information alone. Utilizing the predicted expression, we performed a site-wise analysis and predicted mutations consistent with enhanced developability. The validated assays offer the ability to identify developable proteins at unprecedented scales, reducing the bottleneck of protein commercialization.

Insights into the Organization of the Poxvirus Multicomponent Entry-Fusion Complex from Proximity Analyses in Living Infected Cells.

Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of 11 conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by coimmunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP β-strands. To achieve fluorescence, two additional 20-amino-acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral early/late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and cotransfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quantitative way to determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones were discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of 11 conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.

Improved folding of recombinant protein via co-expression of exogenous chaperones.

Expression of heterologous genes in Escherichia coli is a routine technology for recombinant protein production, but the predictable recovery of properly folded and uniformly bioactive material remains a challenge. Misfolded proteins typically accumulate as insoluble inclusion bodies, and a variety of strategies have been employed in efforts to increase the yield of soluble product. One technique is the overexpression of E. coli protein chaperones during recombinant protein induction, in an effort to increase the folding capacity of the bacterial host. We have developed an alternative approach, by supplementing the host protein folding machinery with chaperones from other species. Extremophiles have evolved under conditions (extremes of temperature, salinity, pressure, and/or pH) that make them attractive candidates for possessing chaperones with novel folding activities. The green fluorescent protein (GFP) of Aequorea victoria, which is predominantly insoluble under typical recombinant expression culture conditions, was employed as an in vivo indicator of protein folding activity for chaperone homologs from a variety of extremophiles. For a subset of the chaperones tested, co-expression with GFP promoted an increase in both fluorescence signal intensity as well as the amount of GFP recovered in the soluble protein fraction. Several archaeal chaperones were also found to be able to refold soluble Lyt_Orn C40 peptidase from inclusion bodies in vitro. In particular, Pf Cpn(MA), a mutant chaperonin which exhibited significant refolding activity, is also shown to deconstruct the morphology and structure of inclusion bodies (Kurouski et al., 2012). Hence, the simple and rapid GFP assay provides a tool to screen for extremophilic chaperones that exhibit folding activity under E. coli growth conditions, and suggests that increasing the repertoire of heterologous chaperones might provide a partial but general solution to the problem of recombinant protein insolubility.

Tomato fruit as a model for tissue-specific gene silencing in crop plants

Use of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated 9)-mediated genome editing has proliferated for use in numerous plant species to modify gene function and expression, usually in the context of either transient or stably inherited genetic alternations. While extremely useful in many applications, modification of some loci yields outcomes detrimental to further experimental evaluation or viability of the target organism. Expression of Cas9 under a promoter conferring gene knockouts in a tissue-specific subset of genomes has been demonstrated in insect and animal models, and recently in Arabidopsis. We developed an in planta GFP (green fluorescent protein) assay system to demonstrate fruit-specific gene editing in tomato using a phosphoenolpyruvate carboxylase 2 gene promoter. We then targeted a SET-domain containing polycomb protein, SlEZ2, previously shown to yield pleiotropic phenotypes when targeted via 35S-driven RNA interference and we were able to characterize fruit phenotypes absent additional developmental perturbations. Tissue-specific gene editing will have applications in assessing function of essential genes otherwise difficult to study via germline modifications and will provide routes to edited genomes in tissues that could not otherwise be recovered when their germline modification perturbs their normal development.

Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava.

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.

Genotypic Regulation of Aflatoxin Accumulation but Not Aspergillus Fungal Growth upon Post-Harvest Infection of Peanut (Arachis hypogaea L.) Seeds.

Aflatoxin contamination is a major economic and food safety concern for the peanut industry that largely could be mitigated by genetic resistance. To screen peanut for aflatoxin resistance, ten genotypes were infected with a green fluorescent protein (GFP)—expressing Aspergillus flavus strain. Percentages of fungal infected area and fungal GFP signal intensity were documented by visual ratings every 8 h for 72 h after inoculation. Significant genotypic differences in fungal growth rates were documented by repeated measures and area under the disease progress curve (AUDPC) analyses. SICIA (Seed Infection Coverage and Intensity Analyzer), an image processing software, was developed to digitize fungal GFP signals. Data from SICIA image analysis confirmed visual rating results validating its utility for quantifying fungal growth. Among the tested peanut genotypes, NC 3033 and GT-C20 supported the lowest and highest fungal growth on the surface of peanut seeds, respectively. Although differential fungal growth was observed on the surface of peanut seeds, total fungal growth in the seeds was not significantly different across genotypes based on a fluorometric GFP assay. Significant differences in aflatoxin B levels were detected across peanut genotypes. ICG 1471 had the lowest aflatoxin level whereas Florida-07 had the highest. Two-year aflatoxin tests under simulated late-season drought also showed that ICG 1471 had reduced aflatoxin production under pre-harvest field conditions. These results suggest that all peanut genotypes support A. flavus fungal growth yet differentially influence aflatoxin production.

Exosome-transmitted microRNA-19a promotes drug resistance in breastcancer by targeting phosphatase and tensin homolog/protein kinase B/p53 axis

Objective To investigate the specific effect and underlying molecular mechansim of microRNA (miRNA, miR)-19a through MCF-7/A secrete dexosomes in drug resistance transfer. Methods Exosomes were extracted from the supernatant of cultured MCF-7/A cells by fractionation ultracentrifugation and were identified by transmission electron microscopy and Nanosight. GFP-MCF-7/S, a breast cancer sensitive cell line stably expressing green fluorescent protein (GFP), was constructed by recombinant lentiviral vector with GFP. The co-cultured experiments of cell-cell and cell-exosomewere designed to observe thephenomenon of drug resistance transfer. Real-time quantitative polymerase chain reaction(Real-time PCR), Western blotting and cell transfection techniquewere used to determine the specific effects of miR-19a and phosphatase and tensin homologue deleted on chromosometen (PTEN)/protein kinase B (Akt)/p53 axis in the progress of drug resistance. Results Compared to MCF-7/S, the expression of miR-19a was significantly increased in MCF-7/A cells (1.03±0.16 vs. 3.56±0.58, P=0.002), down-regulaion of miR-19a apparently increased sensitivity to Adriamycin (ADM) in MCF-7/A cells [(41.17±10.65)% vs. (23.45±8.89)%, P=0.011]. Transmission electron microscopy showed that the exosomes cells had adiameter of 40-100 nm and exhibited round or elliptic shape secreted by both two type cancer cells. The cell inhibition ratio of ADM detected by the intensity of GFP and cell counting kit-8 (CCK-8) assay showed that cell inhibition rate of MCF-7/S+ MCF-7/A and MCF-7/S+ MCF-7/A exos group were significantly lower than the MCF-7/S+ MCF-7/S [(31.50±6.68)% vs. (51.20±12.44)%, P=0.006] and MCF-7/S+ MCF-7/S exos group [(27.67±6.02)% vs. (49.65±10.52)%, P=0.000], which companied with the increasing expression miR-19a (1.01±0.13 vs. 2.67±0.62, P=0.000). Western blotting assay showed that miR-19 in exosomes secreted by drug resistant cancer cells significantly inhibited the expression of PTEN (1.15±0.18 vs. 0.67±0.13, P=0.000) and p53 protein (0.99±0.18 vs. 0.49±0.11, P=0.005), whereas increases phosphorylation of Akt (1.15±0.18 vs. 0.67±0.13, P=0.000), which promoted the occurrence of drug resistance in receptor cells. Conclusion MiR-19a transfers drug resistance between breast cancer cells by exosome secreted from drug-resistance cancer cells through the regulation of PTEN/Akt/p53 axis in receptor cancer cells. Key words: Exosome; MicroRNA-19a; Breast cancer; Drug assistant; Phosphatase and tensin homologue deleted on chromosometen/protein kinase B/p53 axis

A novel small RNA CoaR regulates coenzyme A biosynthesis and tolerance of Synechocystis sp. PCC6803 to 1-butanol possibly via promoter-directed transcriptional silencing

BackgroundMicrobial small RNAs (sRNAs) have been proposed as valuable regulatory elements for optimizing cellular metabolism for industrial purposes. However, little information is currently available on functional relevance of sRNAs to biofuels tolerance in cyanobacteria.ResultsHere, we described the identification and functional characterization of a novel 124 nt sRNA Ncl1460 involved in tolerance to biofuel 1-butanol in Synechocystis sp. PCC 6803. The expression of Ncl1460 was verified by blotting assay and its length was determined through 3′ RACE. Further analysis showed that Ncl1460 was a negative regulator of slr0847 (coaD) and slr0848 operon responsible for coenzyme A (CoA) synthesis possibly via promoter-directed transcriptional silencing mechanisms which has been widely discovered in eukaryote; thus Ncl1460 was designated as CoaR (CoA Biosynthesis Regulatory sRNA). The possible interaction between CoaR and target genes was suggested by CoA quantification and green fluorescent protein assays. Finally, a quantitative proteomics analysis showed that CoaR regulated tolerance to 1-butanol possibly by down-regulating CoA biosynthesis, resulting in a decrease of fatty acid metabolism and energy metabolism.ConclusionsAs the first reported sRNA involved CoA synthesis and 1-butanol tolerance in cyanobacteria, this study provides not only novel insights in regulating mechanisms of essential pathways in cyanobacteria, but also valuable target for biofuels tolerance and productivity modifications.

Differentiation of osteoblast‑like cells and ectopic bone formation induced by bone marrow stem cells transfected with chitosan nanoparticles containing plasmid‑BMP2 sequences.

The present study investigated the efficiency of the use of chitosan nanoparticles containing plasmid‑bone morphogenetic protein2 (pBMP2) sequences (CNPBs) to induce the differentiation of bone marrow stem cells (BMSCs) into osteoblast‑like cells that may be able to promote ectopic bone formation. pBMP2s were constructed, and chitosan nanoparticles were incubated with 50,100or200µg/mlpBMP2. BMSCs were collected from the tibiae and femurs of 6‑week old rats, cultured and treated with the CNPBs or200µg/mlpBMP2 as a positive control. Transfection efficiency was confirmed using the green fluorescent protein assay. Histological staining methods, including alkaline phosphatase, Wright's and von Kasso staining, were used to identify features of osteoblast‑like cells differentiated from BMSCs. Expression levels of the markers of osteoblasts, such as alkaline phosphatase, osteoprotegerin, osteocalcin and osteopontin, were determined to verify the differentiation of BMSCs into osteoblast‑like cells. Ectopic bone formation was observed following the integration of polyglycolic acid (PGA) scaffolds with CNPBs and BMSCs, which were implanted into the dorsal muscles of Sprague‑Dawley rats. Exposure to CNPBs led to the transfection of BMSCs with BMP2. The transfected BMSCs possessed the characteristic phenotypes of osteoblasts. The expression levels of alkaline phosphatase, osteoprotegerin, osteocalcin and osteopontin were significantly higher in the transfected cells compared with the control group, particularly the CBP200 group. PGA scaffolds integrated with BMSCs and CNPBs induced ectopic bone formation, as changes in the morphology of cells were observed using histological staining. Therefore, CNPBs may be a promising method of promoting the formation of novel bone tissue.

Estrogen receptor activation by tobacco smoke condensate in hormonal therapy-resistant breast cancer cells

The relationship between tobacco smoke and breast cancer incidence has been studied for many years, but the effect of smoking on hormonal therapy has not been previously reported. We investigated the effect of smoking on hormonal therapy by performing in vitro experiments. We first prepared tobacco smoke condensate (TSC) and examined its effect on estrogen receptor (ER) activity. The ER activity was analyzed using MCF-7-E10 cells into which the estrogen-responsive element (ERE)-green fluorescent protein (GFP) reporter gene had been stably introduced (GFP assay) and performing an ERE-luciferase assay. TSC significantly activated ERs, and upregulated its endogenous target genes. This activation was inhibited by fulvestrant but more weakly by tamoxifen. These results suggest that the activation mechanism may be different from that for estrogen. Furthermore, using E10 estrogen depletion-resistant cells (EDR cells) established as a hormonal therapy-resistant model showing estrogen-independent ER activity, ER activation and induction of ER target genes were significantly higher following TSC treatment than by estradiol (E2). These responses were much higher than those of the parental E10 cells. In addition, the phosphorylation status of signaling factors (ERK1/2, Akt) and ER in the E10-EDR cells treated with TSC increased. The gene expression profile induced by estrogenic effects of TSC was characterized by microarray analysis. The findings suggested that TSC activates ER by both ligand-dependent and -independent mechanisms. Although TSC constituents will be metabolized in vivo, breast cancer tissues might be exposed for a long period along with hormonal therapy. Tobacco smoke may have a possibility to interfere with hormonal therapy for breast cancer, which may have important implications for the management of therapy.

利用高压水枪法将双报告基因导入小鼠肝脏

目的：通过高压水枪法将双报告基因[即绿色荧光蛋白(GFP)和萤火虫荧光素酶(Luc)]导入小鼠肝脏，以熟练掌握高压水枪法，其将为后续相关研究奠定方法学基础。方法：将质粒pcEF.luc-IRES-GFP瞬时转染入人肝癌细胞SMMC-7721以进行载体体外功能验证，并利用高压水枪法将质粒pcEF.luc-IRES-GFP经尾静脉注射入小鼠肝脏内，经小动物活体成像系统活体检测GFP和Luc信号，同时免疫组化检测GFP在肝组织中的表达情况。结果：瞬转结果显示，7721细胞上可检测到GFP和Luc表达；利用高压水枪法将pcEF.luc-IRES-GFP导入小鼠肝脏后，利用小动物活体成像仪成功在肝内检测到GFP和Luc信号；免疫组化结果显示部分肝细胞表达GFP。结论：成功掌握了将目的基因导入小鼠肝脏的高压水枪法，这为相关后续研究打下良好基础。 Objective: Set up the system for hydrodynamic transfection of double reporter genes [green fluo-rescent protein (GFP) and firefly luciferase (Luc)] into mouse liver. Methods: To in vitro verify the functionality of pcEF.luc-IRES-GFP, human hepatocellular carcinoma 7721 cells were transiently transfected with pcEF.luc-IRES-GFP, followed by GFP and Luc assay. Hydrodynamic transfection was employed to deliver pcEF.luc-IRES-GFP into mice via tail-vein injection, followed by in vivo and ex vivo imaging to detect Luc and GFP. Moreover, immunohistochemistry (IHC) were performed to detect GFP expression in mouse liver cells. Results: The expression of GFP and Luc was detected in 7721 cell transiently transfected by pcEF.luc-IRES-GFP. Luc signals were detected in liver by in vivo bioluminescence imaging, and both Luc and GFP were detected in isolated liver by in vivo bioluminescence and fluorescence imaging. IHC revealed the GFP-positive liver cells of mice hydrodynamically transfected via tail-vein injection of pcEF.luc-IRES-GFP. Conclusions: We successfully deliver double reporter genes (GFP and luciferase) into mouse liver by hydrodynamic transfection, which lays a solid foundation for further research.

Influence of dietary soy isoflavones on immature hamster uterotrophic and Hershberger assays

Abstract To select appropriate diet for hamsters used in the uterotrophic and Hershberger assays two rodent diets were compared: Murigran (Agropol, Poland) and Altromin 7010 (Altromin Spezialfutter GmbH&Co., Germany). The contents of bioactive compounds in feeds were evaluated by liquid chromatography, and their oestrogenic activity by yeast enhanced green fluorescent protein assay. In opposition to Altromin, Murigran contained high amounts (μg/kg) of genistein (765 600) and daidzein (132 000), and the oestrogenic activity of these compounds, expressed as 17β-oestradiol equivalent concentration (EEQ), was found to be 9.54 μg EEQ/kg. In in vivo study, Murigran induced a high degree of oestrogenisation in immature hamsters, and females failed to exhibit a normal uterine response to recommended dose of a model oestrogen agonist 17α-ethinyloestradiol. There was no influence of the diet on the weight of five accessory sex organs (ASO): ventral prostate, seminal vesicles with coagulating glands, levator ani bulbocavernosus muscles, Cowper`s glands, and glans penis of control males. However, the impact on ASO response to model androgen agonist, testosterone propionate was observed. The obtained results provide the evidence that phytooestrogen-rich feed modulates the oestrogenic and androgenic response to chemicals.

Biocompatible polyethylenimine-graft-dextran catiomer for highly efficient gene delivery assisted by a nuclear targeting ligand

The aim of the present work was to design a targeting polysaccharide-based cationic polymer for gene delivery. PEI is one of the most widely studied cationic polymers in terms of gene delivery, yet its applications are limited by its inherent drawbacks. To address these drawbacks, the grafted low-molecular-weight branched polyethylenimine (PEI) (1800 Da and 800 Da) with the low cytotoxicity and targeting ligand were integrated into a polymeric system based on biodegradable and biocompatible polysaccharide dextran, to afford a highly efficient gene delivery system. The structure of the grafted polymer (Dextran-g-PEI) was confirmed by nuclear magnetic resonance (NMR). DNA complexation resulted in nanoscale and spherical particles (Dextran-g-PEI/DNA), revealed by transmission electron microscopy (TEM). The sizes of the complexes decreased while the zeta potentials increased with the elevation of the w/w ratio. The agarose gel electrophoresis suggested that complexation process alleviates the DNA degradation by DNase I. Very low cytotoxicity was shown on HeLa, 293T and HepG2 cell lines using MTT cell relative viability assay, a red blood cell aggregation assay as well as hemolytic activity determination, whereas transfection efficiency in vitro of Dextran-g-PEI/DNA complexes was observed by green fluorescent protein assay in 293T, HeLa and HepG2 cells. Cy™3 was used as a molecular probe to evaluate the effect of nuclear targeting ligand (PV7) addition on the cellular uptake of complexes. After the intravenous injection of pEGFP to BALB/c mice xenograft models for transfection in vivo, the expression of green fluorescent protein was observed in the transplanted carcinoma and some of the important organs, such as liver, spleen, kidney and lung. To prove the tumor localization of the polyplexes, the in vivo circulation and biodistribution of the polyplexes were monitored by in vivo fluorescent imaging. Thus it was concluded that Dextran-g-PEI is a potential candidate for effective and biocompatible gene delivery systems.

Development of a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens

Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay (“BlueScreen HC”), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.

CUT-AND-PASTE GFP

A NEW IN VITRO METHOD for splitting and reassembling green fluorescent protein (GFP) gives scientists synthetic control of the composition of this common fluorescent tag. The method will not only help scientists better understand the workings of GFP but also inspire the design of improved proteins for fluorescent labeling in cells. GFP consists of β-strands that form a barrel and a chromophore-containing α-helix inside the barrel. In the new method, GFP is cut at predetermined sites, and to reassemble the protein, the parts removed by the cuts are replaced with synthetic components. The method was inspired by so-called split GFP assays, which are widely used to detect protein interactions in vivo. In these assays, GFP is expressed as two pieces, each fused to proteins whose interaction is being probed in cells. When the proteins interact, GFP forms from the two pieces, and the assembly fluoresces. Although studying the properties of these smaller pieces of GFP ...

A Mechanistically Relevant Cytotoxicity Assay Based on the Detection of Cellular GFP

Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.

A new time-saving transformation system for Brassica napus

By optimizing different parameters for infection, an efficient Agrobacterium-mediated transformation system for Brassica napus was developed. A series of combinations of optical densities, infection durations, concentrations of acetosyringone and silwet L77 were evaluated. Maximum transformation efficiency reached 18.93% when cotyledonary nodes were infected for 10 min with an Agrobacterium suspension of 0.8 OD and 200 iM acetosyingone and 0.02% silwet L77 in the culture medium. In addition, the concentrations of 6-benzyladenine and naphthalene acetic acid were also optimized. Meanwhile, maximum transformation efficiency for green plantlet was 94.38% when cotyledonary nodes were cultivated in the medium containing 4 mg/L 6-benzyladenine and 0.04 mg/L naphthalene acetic after co-cultivation. Transformation was confirmed by polymerase chain reaction analysis, southern blotting analysis and green fluorescent protein assay.