Evaluation of parameters affecting Agrobacterium-mediated transient expression in citrus

Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.

Synthesis and Characterization of A Small Molecule CFTR Chloride Channel Inhibitor

A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.

A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.

Expression of vascular endothelial growth fac-tor gene in primary cultured rat hepatocytes

BACKGROUND: It is the key point for vascular endothelialgrowth factor (VEGF121) gene related therapy as to how totransfect and express the gene safely, effectively and repeat-edly. This study was designed to investigate the VEGF121transfection and expression in primary cultured rat hepato-cyte.METHODS: After construction of vector internal ribosomeentry site-enhanced yellow fluorescent protein (pIRES-EY-FP)/VEGF121, the transfection and expression of the exoge-nous VEGF121 gene in primary cultured rat hepatocyteswere observed through RT-PCR, Western blot and fluores-cent microscopy.RESULTS: pIRES-EYFP/VEGF121 plasmid was construct-ed and transfected successfully into primary cultured rathepatocytes, the transfection and expression of gene in pri-mary cultured rat hepatocytes were examined by RT-PCRand Western blot, and yellow-green fluorescence was ob-served through a fluorescent microscope.CONCLUSION: The successful transfection and expressionof plasmid pIRES-EYFP/VEGF121 in primary cultured rathepatocytes provides a foundation for hepatocyte transplan-tation and gene therapy after modification of hepatocytesby the gene.

Construction of WCB-11:A novel phiYFP arsenic-resistant whole-cell biosensor

The prediction and assessment of environmental pollution by arsenic are important preconditions of advocating environmental protection and human health risk assessment. A yellow fluorescent protein-based whole-cell biosensor for the detection of arsenite and arsenate was constructed and tested. An arsenic-resistant promoter and the regulatory gene arsR were obtained by PCR from the genome ofEscherichia coli DH5ct, andphiYFP was introduced into E. coli DH5ct as a reporter gene to construct an arsenic-resistant whole-cell biosensor （WCB-11） in which phiYFP was expressed well for the first time. Experimental results demonstrated that the biosensor has a good response to arsenic and the expression ofphiYFP. When strain WCB-11 was exposed to As^3＋ and As^5＋, the expression of yellow fluorescence was time-dependent and dose-dependent. This engineered construct is expected to become established as an inexpensive and convenient method for the detection of arsenic in the field.

Ultra-High Field Diffusion MRI Reveals Early Axonal Pathology in Spinal Cord of ALS mice

Background:Amyotrophic lateral sclerosis(ALS)is a disease characterized by a progressive degeneration of motor neurons leading to paralysis.Our previous MRI diffusion tensor imaging studies detected early white matter changes in the spinal cords of mice carrying the G93A-SOD1 mutation.Here,we extend those studies using ultra-high field MRI(17.6 T)and fluorescent microscopy to investigate the appearance of early structural and connectivity changes in the spinal cords of ALS mice.Methods:The spinal cords from presymptomatic and symptomatic mice(80 to 120 days of age)were scanned(ex-vivo)using diffusion-weighted MRI.The fractional anisotropy(FA),axial(AD)and radial(RD)diffusivities were calculated for axial slices from the thoracic,cervical and lumbar regions of the spinal cords.The diffusion parameters were compared with fluorescence microscopy and membrane cellular markers from the same tissue regions.Results:At early stages of the disease(day 80)in the lumbar region,we found,a 19% decrease in FA,a 9% decrease in AD and a 35% increase in RD.Similar changes were observed in cervical and thoracic spinal cord regions.Differences between control and ALS mice groups at the symptomatic stages(day 120)were larger.Quantitative fluorescence microscopy at 80 days,demonstrated a 22% reduction in axonal area and a 22% increase in axonal density.Tractography and quantitative connectome analyses measured by edge weights showed a 52%decrease in the lumbar regions of the spinal cords of this ALS mice group.A significant increase in ADC(23.3%)in the ALS mice group was related to an increase in aquaporin markers.Conclusions:These findings suggest that the combination of ultra-high field diffusion MRI with fluorescent ALS mice reporters is a useful approach to detect and characterize presymptomatic white matter micro-ultrastructural changes and axonal connectivity anomalies in ALS.