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Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.
Subject(s)
Dependovirus , Glial Cell Line-Derived Neurotrophic Factor , HEK293 Cells , Recombination, Genetic , Transduction, Genetic , Cell Line , Polymerase Chain Reaction , Green Fluorescent Proteins , Genetic Vectors , Microscopy, FluorescenceABSTRACT
Aim The limited transfection efficiency for plasmid in primary neonatal rat cardiomyocytes,which are terminal differentiated cells,and long foreign DNA (the RIP140 gene sequence are as long as 3.5 kb) cause us to choose a better system to study RIP140 gene expression in primary non-replicative cells. Methods Full-length of RIP140 was cloned into pAdTracker-CMV shuttle vector,and then recombined with virus backbone pAdEasy-1 vector in BJ5183 bac-teria.Positive recombinant plasmid was confirmed by sequence analysis and restriction enzyme determina-tion,and then transfected into AD293 cells for amplifi-cation.Titers of virus particles were determined by Tis-sue Culture Infectious Dose 50 (TCID50 )method and cell vitality was analyzed by CCK-8 kit in cardiomyo-cytes.RIP140 gene was identified by Western blot. Results Sequence analysis suggested that full-length RIP140 gene was cloned correctly into AdEasyTM sys-tem.Virus titers of Ad-RIP140 and Ad-GFP were 1011.3 and 1011.7 PFU·mL-1 ,respectively.Cell vitali-ty was not affected when the Multiplicity of Infection (MOI)was lower than 200.Green fluorescent protein (GFP)and Western blot analysis showed RIP140 gene was remarkably increased in cardiomyocytes for 12h in-fection by Ad-RIP140 (P<0.05 ).Conclusion Re-combinant adenovirus containing RIP140 gene was suc-cessfully constructed and effectively expressed in car-diomyocytes.These will be helpful for further research on the function of RIP140 in cardiomyocytes.
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Objective To optimize multiplicity of infection ( MOI) and antibiotics ( blasticidin) concentration selecting BSD gene in construction of monoclonal stable cell line by lentivirus vector-mediated RNA interence silenced gene SGMS2.Methods The INS-1 cells were transfected by fluorescence labeled negative control SGMS2-siRNA lentivirus at MOI of 0, 10, 30, 60 and 120 TU number/cell.The cells were photographed under fluorescent microscopy after 72 h cultivation, then fluorescence ratio and apoptosis rate were calculated to determine optimal MOI.The INS-1 cells were treated by blasticidin with different concentrations of 0, 1, 2, and 3 μg/mL, and the apoptosis rate was observed to acquire optimal concentration of antibiotics.The INS-1 cells were transfected by negative control SGMS2-siRNA lentivirus and SGMS2-siRNA lentivirus (virus titer:1 ×108TU/mL) at optimal MOI and positive-transfected cells were selected by blasticidin at optimal concentration, then mixed cell lines were acquired.The monoclonal cell line was constructed at fluorescence ratio of 90%.Results The optimal MOI was 60 with 100% fluorescence ratio, less than 0.5% apoptosis rate and keep original cellular morphology.The optimal concentration of blasticidin was 2 μg/mL with cell adherence disappear and all cells apoptosis.The Ct value of INS-1-SEMS2 cells detected at the second time was 28.21, which was greater than 27.58 at the first time.The interfering efficiency of siRNA was 77.78% which indicated a successful expression of siRNA and construction of monoclonal stable cell line ( INS-1-SEMS2 ).Conclusion The monoclonal stable cell line was successfully constructed by lentivirus vector-mediated RNA interence silenced gene SGMS2.
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Background: \r\n', u'Rotavirus is the major important cause of infectious infantile gastroenteritis and diarrhea, especially the children aged 6 months to 24 months. Some efforts made to provide safe water and cultivate the hygiene have not reduced the diarrhea caused by rotaviruses. Develope a vaccine agaisnt this virus is of high importance. A question about the production of this kind of vaccine targeted athow to make human rotaviruses strains adapt cultured cells.\r\n', u'Objectives\r\n', u'To determine the multiplicity of infection (MOI) of human rotaviruses strains in vero cell.\r\n', u'Subjects and method: \r\n', u'The multiplicity of infection of Human rotaviruses strains (G1 P8, G1P4, and G4P6) were selected and the CDC- Atlanta US supplied the vero cell.Use vero cells cultivate, temper the vero cell and, applying direct immunofluorescence to determine the multiplicity of G1P8, G1P4, C4P6.\r\n', u'Results:\r\n', u'The MOI of G1P8, G1P4 and C4P6 are 0.02 ffu/cell, 0.15ffu/cell, and 0.35 ffu/cell, respectively.\r\n', u'Conclusion:\r\n', u'Using the vero cell to multiple the rotaviruses is the effective part during the production of vaccines against rotaviruses. The MOI of human rotaviruses strains G1P8, G1P4 and G4P6 are in order:0,02 ffu/cell, 0,15ffu/cell, and 0,35% ffu/cell. \r\n', u'
Subject(s)
Rotavirus , Vero Cells , Cell Culture TechniquesABSTRACT
OBJECTIVE To comprehend the main pathogens and their drug resistance of multiple organ dysfunction syndrome(MODS) patients in ICU.METHODS We retrospectively analyzed all the bacteria isolated from 40 MODS patients in ICU.RESULTS The number of bacteria strains isolated was 173,92 G-bacteria strains made up 53.18%,60 G+ bacteria strains made up 34.68%,and 21 fungi strains made up 12.14%.The top six were Staphylococcus aureus(23.70%,MRSA was 13.87%),Pseudomonas aeruginosa(14.45%),Acinetobacter baumannii(11.56%),Stenotrophomonas maltophilia(8.67%),Candida tropicalis(8.09%),and Enterococcus faecalis(7.51%).The susceptive rate of S.aureus and Enterococcus to vancomycin was all 100%,the susceptive rate of A.baumannii and Klebsiella pneumoniae to carbapenems was high.64% patients had the multiplicity of infection(MOI) which always linked with long period in ICU,respiratory failure and mechanical ventilation.CONCLUSIONS MODS patients have a high morbility of G+ bacteria,fungi and MOI,most pathogens show multi-resistance to commonly used antibiotics.Strengthening the monitoring of infection and reasonable using antibiotics should be taken.
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OBJECTIVE To characterize bacteriophage isolated from sewage with indicator of Klebsiella pneumoniae subsp pneumoniae.METHODS Bacteriophage was isolated from sewage by double-layer agar plate method,identified lytic or lysogenic capacity by induced methods of ultraviolet ray and mitomycin C,performed one-step growth experiments,decided the optimal multiplicity of infection,and its structure was observed with electron microscope.RESULTS The study found a strain of bacteriophage against the K.pneumoniae subsp pneumoniae.The phage had a head of 180 nm in diameter and a tail of 210 nm in length.The plaque was transparent and 4-7 mm in diameter.the optimal multiplicity of infection was 4,latent period was 35 min and burst period was 40 min,the average burst size was about 94 PFU/cell.CONCLUSIONS The isolated bacteriophage belongs to Stylovinidae,and it is a lytic bacteriophage with narrow host spectrum.Moreover,it is only sensitive to a part of K.pneumoniae subsp pneumoniae and a few of Escherichia coli strains.
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Objective To identify some basic biological properties,optimal multiplicity of infection(MOI),one-step growth kinetics,serum neutralization rate constant of Pseudomonas aeruginosa phage PaP3 were determined, respectively. Methods The host bacteria were infected with PaP3 at six different MOI(0.0001,0.001,0.01,0.1,1 and 10,then lysated adequately, the phage titer of supernatant was determined.For one-step growth experiments,the host bacteria were infected with PaP3 at MOI=10, following centrifugation after 15 min adsorption,the pellet containing (partially) infected cells was resuspended in 3 ml of pre-warmed LB broth and incubated with 160rpm at 37℃.Samples(50?l) were taken at 10 min-intervals and immediately tittered by the double-layer agar plate method. A rabbit was immunized with purified PaP3 to prepare anti-PaP3 serum, reaction constants between antiserum and PaP1, PaP2, PaP3 were determined using cross neutralization test. Results 0.001 MOI-infected host bacteria gave the highest phage offsprings. One-step growth curve was determined according to the results of one-step growth experiments.Reaction constant was determined using neutralization test. Conclusion The optimal MOI of PaP3 was determined to be 0.001. One-step growth curve for PaP3 shows that the latent period is about 20 min, the rise period is 60 min, and the average burst size is about 31pfu/cell. Rate constant of reaction between antiserum and PaP3 is 262.