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

In order to optimize the conditions of construction BAC library, the transformation efficiency of E. coli DH10B was studied in this paper. Our data prove much higher competence of electroporation (reaches 2.19 x 10(10) cfu/microg pUC19 DNA) when harvesting the cells between an OD550 of 0.7 - 0.8. Five different electric field strength (from 9 kV/cm to 25 kV/cm) and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCLD04541 DNA, as well as three bacterial artificial chromosomes (BACs) ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.
Chromosomes, Artificial, Bacterial ; genetics ; DNA, Bacterial ; chemistry ; genetics ; Electroporation ; methods ; Escherichia coli ; genetics ; Molecular Weight ; Plasmids ; genetics ; Transformation, Genetic

A novel transformation method was firstly established using glass beads in Dunaliella salina (D. salina). The results showed that the GUS gene, a reporter gene, was successfully expressed in D. salina. Cells of D. salina presented blue color under the microscope after stained. In addition, different factors which influenced transformation were optimized including the transformation consecutive time, rotate speed, concentration of the plasmid and PEG 6000. The experiment indicated that this fit together can obtain the best results for D. salina transformation: adding 150 microL PEG and 90 microL plasmid DNA to 800 microL culture of D. salina (10(6) cells/mL) containing 300 mg glass beads, swirling 12 seconds under the rotate speed 2400r/min. This newly method can be used as a potential tool in the research of D. salina gene engineering with the advantage of more simpleness, convenience, quickness and less expense.
Chlorophyta ; genetics ; DNA ; chemistry ; genetics ; Genetic Engineering ; methods ; Glass ; Glucuronidase ; genetics ; metabolism ; Histocytochemistry ; Microspheres ; Plasmids ; genetics ; Polyethylene Glycols ; chemistry ; Time Factors ; Transformation, Genetic ; genetics

In order to establish K562 line with stable expressions of hermap and hermap-siRNA, amplified hermap and hermap-siRNA were cloned into pEGFP-c1 and pRNAT to acquire hermap-pEGFP-c1 and hermap-siRNA-pRNAT, respectively. These two plasmids were electrotransferred into K562 cells, then were followed by culturing with G418. The result showed that the transfer rate of hermap-pEGFP-c1-K562 and hermap-siRNA-pRNAT-K562 plasmids were 10.0% and 9.3%, respectively. After selective culture by G418, these two cell lines were still able to express GFP. It is concluded that the eukaryotic expression plasmids containing hermap and hermap-siRNA have been successfully constructed, and the cell lines of hermap-K562 and hermap-siRNA-K562 are established, definitely contributing to further functional investigation on HERMAP and its interaction with other proteins.
Erythrocytes ; chemistry ; Gene Silencing ; Humans ; K562 Cells ; Membrane Proteins ; genetics ; Plasmids ; RNA, Small Interfering ; Transfection

This study inquired about the influences on the gene delivery efficiency of polyethylenimine. pSVbeta plasmids were transferred into COS-7 and NIH3T3 cells with polyethylenimine. Influences of plasmids factor, albumin, serum, cell density, operation methods and polyethylenimine/DNA preserve factors on transfection efficiency were investigated. Inhibitors of biological activities in plasmids could be removed by ultrafiltration with cutoff molecular weight of 3000 or 10000. Linear plasmids lowered transfection efficiency. Serum and albumin in the culture medium decreased the transfection efficiency of polyethylenimine/DNA. Cell density was associated with PEI/DNA transfection efficiency. Incubation of PEI/DNA complexes with the cells for 8 h and then aspiration for removal of the complexes could obtain an optimal transfection effect. Freezing of PEI/DNA complexes significantly decreased transfection efficiency. In conclusion, polyethylenimine could obtain optimal and reduplicate transfection results by controlling related factors.
3T3 Cells ; Animals ; COS Cells ; Cercopithecus aethiops ; DNA ; genetics ; Genetic Vectors ; genetics ; Mice ; Plasmids ; genetics ; Polyethyleneimine ; chemistry ; Transfection ; Ultrafiltration

Thermostable alpha-amylase from Pyrococcus furious is an important industrial enzyme in brewing and alcohol production. Eexpression of the thermostable a-amylase in plants can reduce greatly costs in the production of alcohol using crop plants. A chloroplast expression vector, p64A, containing the thermostable alpha-amylase gene from Pyrococcus furious, was constructed with clpP-trnL-petB-chlL-rp123-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spetinomycin-resistant aadA gene as select marker. The plasmid p64A was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Nine independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the transgene and cultivation in the dark all showed that the a-amylase gene had been integrated into chloroplast genome of C. reinhardtii. The activity of amylase expressed in the chloroplast of C. reinhardtii was detected by amylase activity assay and found to be as high as 77.5 u/g fresh weight of cells. These experimental results demonstrated the possibility of using transgenic chloroplasts of plant as bioreactors for production of industrial enzymes.
Chlamydomonas reinhardtii ; genetics ; Chloroplasts ; genetics ; Enzyme Stability ; Plasmids ; Polymerase Chain Reaction ; Pyrococcus furiosus ; enzymology ; alpha-Amylases ; chemistry ; genetics ; metabolism

To explore the role of the mutations G38R and D40G of Annexin A11 (ANXA11) in the onset of amyotrophic lateral sclerosis (ALS).
 Methods: The plasmids expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were constructed, respectively. The recombinant plasmids were then transfected into HEK293 cells respectively followed by cycloheximide (CHX) treatment for 0, 2, 4 and 8 h. The protein expressions of ANXA11 wild type, ANXA11 G38R and ANXA11 D40G mutations were determined by Western blot. Gray analysis by Image J was performed to compare the half-life of each protein. The NSC-34 cell lines constantly expressing ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein were established. The cells were treated with NP-40 lysis buffer to examine the protein solubility by Western blot.
 Results: Both ANXA11 G38R protein and ANXA11 D40G protein showed a shorter half-life than ANXA11 wild type protein (P<0.05), while there was no difference between ANXA11 G38R protein and ANXA11 D40G protein (P>0.05). There was no visible insoluble substance in the NP-40 lysates for ANXA11 wild type protein, ANXA11 G38R protein and ANXA11 D40G protein.
 Conclusion: G38R and D40G mutations reduce the stability of ANXA11 protein. G38R and D40G mutations do not alter ANXA11 solubility.
Amyotrophic Lateral Sclerosis ; genetics ; metabolism ; Annexins ; chemistry ; genetics ; metabolism ; HEK293 Cells ; Humans ; Mutation ; Plasmids ; genetics ; Protein Stability ; Solubility ; Transfection

A novel transferrin modified non-viral gene delivery system Tf-PLPD was developed and the related characteristics was investigated. Blank procationic liposomes were prepared by film dispersion-filteration method. PLPD was prepared as follows by first mixing the plasmid DNA and protamine together, then the resulted polyplexes were incubated for 10 min at room temperature, followed by addition of preformed blank procationic liposomes. Transferrin was adsorbed at the surface of PLPD via electrostatic interactions to form Tf-PLPD. Central composite design (CCD) was employed to optimize the formulation. The HepG2 cells were transfected using lacZ as reporter gene and characteristics such as the morphology, the mean particle size, the zeta potential and the transfection efficiency in HepG2 cells were further investigated by different methods. The resulting PLPD had a regular spherical surface with an average size of (228. 9 +/- 8. 0) nm (polydispersity index, PDI = 0. 122 +/- 0. 02, n = 3) , a zeta potential of ( - 25. 08 +/-2. 50) mV (n = 3) and a transfection efficiency of (12. 18 +/- 3. 80) mU x mg(-1) (protein). The Tf-PLPD had an average size of (240 +/- 12) nm (polydispersity index, PDI = 0. 150 +/- 0. 03, n = 3), a zeta potential of ( - 24. 10 +/- 2. 50) mV ( n = 3) and a transfection efficiency of (24. 26 +/- 2. 60) mU x mg(-1) (protein) , 20 times greater than that of the naked plasmid DNA. The presence of serum didn' t affect the tansfection activity of PLPD or Tf-PLPD. Compared to one kind of cationic liposomes (liposome-protamine-DNA, LPD), the PLPD and Tf-PLPD had much less cytotoxicity to three hepatic cell lines (including HepG2, SMMC7721 and Chang' s normal hepatocyte). The results indicated that the Tf-PLPD is a perspective non-viral vector for gene delivery systems.
Cations ; chemistry ; Cell Line ; Cell Line, Tumor ; Cell Survival ; DNA ; chemistry ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Liposomes ; chemistry ; Liver Neoplasms ; genetics ; pathology ; Particle Size ; Plasmids ; chemistry ; genetics ; Protamines ; chemistry ; Transfection ; methods ; Transferrin ; chemistry ; genetics

Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 microg, 50 EU and 10 microg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75-1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Cetrimonium Compounds ; chemistry ; Chemical Precipitation ; DNA ; genetics ; isolation & purification ; DNA, Bacterial ; genetics ; isolation & purification ; Escherichia coli ; chemistry ; genetics ; Plasmids ; genetics ; isolation & purification

Protein disulfide isomerase-related protein A (PRPA) was highly expressed (about 34%) in Escherichia coli by inserting the whole PRPA cDNA into the vector pET23b. After expression, the purified protein was acquired through ammonium fractional precipitation and Bio-Rex 70 chromatography. PRPA shows low disulfide isomerase activity (only about 1/250 of that of hPDI), decreases the reactivation yield of denatured and reduced lysozyme either in redox and non-redox Hepes buffer or redox PBS buffer and facilitates the aggregation of denatured and reduced lysozyme. Fluorescence spectra of PRPA indicate that PRPA has more hydrophobic groups at surface than that of hPDI, and which can be used to explain why PRPA has anti-chaperone activity during the refolding of denatured and reduced lysozyme.
Cloning, Molecular ; Fungal Proteins ; chemistry ; genetics ; isolation & purification ; Muramidase ; chemistry ; Plasmids ; Protein Folding ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Spectrometry, Fluorescence