The preparation and cell transfection of chitosan-DNA nanoparticles were studied. The TFPI (tissue factor pathway inhibitor) or EGFP (enhanced green fluorescent protein) plasmid DNA was encapsulated with chitosan to form gene nanoparticles. The results with TEM showed that the nanoparticles were of sphere shape. The mean diameter of the nanoparticles was 149 nm and the diameter ranged from 80-250 nm, which were measured by the photo related spectrometry (PCS). The encapsulation efficiency of DNA was 96% +/- 1.38% and the DNA content in the nanoparticles was 37% +/- 3.0%. The encapsulated DNA could be protected from the degradation by DNase I. The transfection efficiency of chitosan nanoparticles were about equivalent to that of the LipofectAMINETM reagent. Our results also showed that chitosan nanoparticles were nontoxic to cultured cells.
Cells, Cultured ; Chitosan ; chemistry ; DNA ; chemistry ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lipoproteins ; genetics ; Muscle, Smooth, Vascular ; chemistry ; metabolism ; Nanoparticles ; chemistry ; Plasmids ; genetics ; Transfection

Rat calcineurin (CaN) A alpha isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EGFP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A alpha (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.
Adenoviridae/*genetics ; Calcineurin/*biosynthesis ; Calcineurin/genetics ; Cloning, Molecular ; DNA, Complementary/genetics ; Genetic Vectors/genetics ; Green Fluorescent Proteins/biosynthesis ; Green Fluorescent Proteins/genetics ; Myocardial Reperfusion Injury/*genetics ; Myocardium/chemistry ; Rats, Wistar ; Recombination, Genetic/genetics

In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E. coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.
Gene Products, tat ; chemistry ; metabolism ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Microscopy, Fluorescence ; Multiple Myeloma ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; metabolism ; Tumor Cells, Cultured

The objective of this study is to explore the application possibility of chitosan/pcDNA-EGFP-TGFPbeta1 nanoparticles in the transfection of synovial-derived mesenchymal stem cells (SDMSCs). Chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles were fabricated through method of ionic crosslinking. The SDMSCs were harvested from rabbit joints and cultured to passage 3. The SDMSCs were then transfected with chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles. Scanning electronic microscope (SEM) was employed to detect the shape and diameter of the nanoparticles. The transfected SDMSCs were examined under the fluorescence microscope and detected through the flow cytometry (FCM). The SEM examination showed that the contour of the fabricated chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles was round and its average diameter was 50 nm. After being cultured for 48 h, the SDMSCs transfected by chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles could be detected under the fluorescence microscope, and the live SDMSCs could also be examined through FCM. The transfection rate was 8% - 10%. Therefore, it suggested that the chitosan/pcDNA-EGFP-TGFbeta1 nanoparticles fabricated through the method of ionic crosslinking could transfect the SDMSCs, but the transfection rate should be improved.
Animals ; Chitosan ; chemistry ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Mesenchymal Stromal Cells ; cytology ; Microscopy, Electron, Transmission ; Microscopy, Fluorescence ; Nanoparticles ; chemistry ; Rabbits ; Transfection ; Transforming Growth Factor beta1 ; genetics

To evaluate the feasibility of using polyethyleneimine (PEI) coated magnetic iron.oxide nanoparticles (polyMAG-1000) as gene vectors. The surface characteristics of the nanoparticles were observed with scanning electron microscopy. The ability of the nanoparticles to combine with and protect DNA was investigated at different PH values after polyMAG-1000 and DNA were combined in different ratios. The nanoparticles were tested as gene vectors with in vitro transfection models. Under the scanning electron microscope the nanoparticles were about 100 nm in diameter. The nanoparticles could bind and condense DNA under acid, neutral and alkaline conditions, and they could transfer genes into cells and express green fluorescent proteins (GFP). The transfection efficiency was highest (51%) when the ratio of nanoparticles to DNA was 1:1 (v:w). In that ratio, the difference in transfection efficiency was marked depending on whether a magnetic field was present or not: about 10% when it was absent but 51% when it was present. The magnetic iron oxide nanoparticles coated with PEI may potentially be used as gene vectors.
Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Ferric Compounds ; chemistry ; metabolism ; Gene Targeting ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Magnetics ; Nanotechnology ; Particle Size ; Polyethyleneimine ; chemistry ; Transfection ; methods

Small interference RNA (siRNA) induced RNA interference (RNAi) technology has shown high specificity and high efficiency of silencing target gene expression, and it is becoming a promising candidate drug for the therapy of cancer and viral infection diseases. At present, the lack of safe and effective carrier materials and delivery systems of siRNA through extracellular and intracellular barriers still hampers the clinical application. In order to overcome this difficulty, we proposed using chitosan, naturally occurring polycation, to form complex siRNA against green fluorescence protein (siRNA-eGFP). The spherical and stable chitosan-siRNA nanoparticles with 83%-94% siRNA complex efficiency can be formulated under mild electrostatic interaction. The size and Zeta potential of nanoparticles were within the range of 90-180 nm and 10-30 mV, respectively. 80% cell viability could be maintained inthe course of incubating with chitosan-siRNA nanoparticles. Moreover, nearly 80% gene silencing efficiency of chitosan-siRNA nanoparticles was realized.
Cell Line ; Chitosan ; pharmacology ; Drug Carriers ; chemistry ; Drug Delivery Systems ; Green Fluorescent Proteins ; chemistry ; Humans ; Nanoparticles ; RNA Interference ; RNA, Small Interfering ; genetics ; pharmacology

OBJECTIVETo explore the mechanisms by which HFBI fusions increase recombinant fusion protein accumulation in plants.

METHODSThe HFBI sequence from Trichoderma reesei was synthesized and two plant expression vectors for expression of green fluorescence protein (GFP) and GFP-HFBI were constructed. The vectors were inoculated in Nicotiana benthamiana plants through agroinfiltration, and the expression levels and mRNA accumulation levels of GFP in Nicotiana leaves were examined by Western blotting, ELISA and RT-PCR.

RESULTSThe HFBI fusion tag significantly enhanced the accumulation of GFP in the leaves of N. benthamiana without causing toxic effects. Endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of spherical protein particles in the plant cells.

CONCLUSIONHFBI fusions can increase the accumulation of its fusion partner in plants by forming stable protein particles, which probably shields the target protein from endogenous protease-induced degadation. HFBI fusion technology provides an alternative to improving recombinant protein expression in plants from agroinfection-compatible expression vectors.

Endoplasmic Reticulum ; Genetic Engineering ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; Imidazoles ; chemistry ; Plant Leaves ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Tobacco ; genetics ; metabolism

Cell-free protein expression system is a new method to express target protein in vitro and has been widely applied to the study of protein structure, protein function and other related fields. Preparation of cell extract is one of the key factors that affect the efficiency of the cell-free system. To improve the efficiency and economical feasibility of cell-free protein synthesis, we discussed the parameters during the preparation of the cell extract. These parameters include centrifugation speed, pre-incubation, and dialysis. We used the green fluorescent protein as the reporter protein, and obtained a simple procedure for the preparation of Escherichia coli cell extract. A simple centrifugation step (12 000 x g, 10 min) followed by a brief incubation was sufficient for the preparation of an active cell extract to support protein expression with higher productivity (209 microg/mL). Compared to the traditional E. coli S30 procedure, the processing time was reduced by 62%, and the productivity was increased by 2.6 times. The new procedure will make the advantage of cell-free technology more obvious, and promote its wider application.
Cell Fractionation ; methods ; Cell-Free System ; Escherichia coli ; cytology ; genetics ; metabolism ; Escherichia coli Proteins ; biosynthesis ; chemistry ; isolation & purification ; Green Fluorescent Proteins ; metabolism

Chloride channels belong to a superfamily of ion channels that permit passive passage of anions, mainly chloride, across cell membrane. They play a variety of important physiological roles in regulation of cytosolic pH, cell volume homeostasis, organic solute transport, cell migration, cell proliferation, and differentiation. However, little is known about the functional regulation of these channels. In this study, we generated an integrated transgenic worm strain expressing green fluorescence protein (GFP) fused CLC-type chloride channel 1 (CLH-1::GFP), a voltage-gated chloride channel in Caenorhabditis elegans (C. elegans). CLH-1::GFP was expressed in some unidentified head neurons and posterior intestinal cells of C. elegans. Interacting proteins of CLH-1::GFP were purified by GFP-Trap, a novel system for efficient isolation of GFP fusion proteins and their interacting factors. Mass spectrometry (MS) analysis revealed that a total of 27 high probability interacting proteins were co-trapped with CLHp-1::GFP. Biochemical evidence showed that eukaryotic translation elongation factor 1 (EEF-1), one of these co-trapped proteins identified by MS, physically interacted with CLH-1, in consistent with GFP-Trap experiments. Further immunostaining data revealed that the protein level of CLH-1 was significantly increased upon co-expression with EEF-1. These results suggest that the combination of GFP-Trap purification with MS is an excellent tool to identify novel interacting proteins of voltage-gated chloride channels in C. elegans. Our data also show that EEF-1 is a regulator of voltage-gated chloride channel CLH-1.
Animals ; Animals, Genetically Modified ; Caenorhabditis elegans ; genetics ; metabolism ; Caenorhabditis elegans Proteins ; metabolism ; Chloride Channels ; metabolism ; Green Fluorescent Proteins ; chemistry ; Mass Spectrometry ; Peptide Elongation Factor 1 ; metabolism

Abstract:By using PVX derived vector pGR107, the effect of BYDV-MP nuclear localization signal on the movement of PVX was studied. BYDV-MP was cloned into pGR107 using GFP as an indicator. BYDV-MP was then shown to induce the systemic infection and exacerbate the symptom of PVX through infecting Nicotiana benthamiana. When the PVX gene encoding 25kD protein, which functioned as a systematic movemnet protein,was deleted and the above experiment was repeated, the result showed that BYDV-MP could compensate the systemic movement of PVX. A serial mutants with substitutions on the fifth, sixth and seventh amino acids of BYDV-MP nuclear localization signal was further constructed. It was found that the mutants at the fifth, sixth amino acids in BYDV-MP nuclear localization signal could only delay or weaken systemic movement of PVX whereas the mutant at seventh amino acid could entirely inhibit systemic movement of PVX.
Amino Acid Sequence ; Green Fluorescent Proteins ; genetics ; Luteovirus ; physiology ; Molecular Sequence Data ; Nuclear Localization Signals ; chemistry ; physiology ; Plant Viral Movement Proteins ; physiology ; Potexvirus ; genetics ; physiology