Construction,Protein Expression,and Subcellular Localization of Various Kinase Mutant Plasmids of GATA1 / 中国医科大学学报

Objective The GATA1 mutant GATA1 S161A S187A (death type) and GATA1 S161D S187D (activated) eukaryotic expression vectors were constructed using the large primer method,and,to explore their biological function and potential tumor treatment targets,the expression and localization of the fusion protein in cells were confirmed. Methods S161A,S187A,S161D,and S187D mutants were amplified by GFP-GATA1 WT,which served as the template. The recombinant plasmid was cloned into a pEGFP-C1 expression vector and transfected into HEK293 cells by immunoblotting expression of the fusion protein. Results The eukaryotic expression vectors pEGFP-GATA1 S161A S187A and pEGFP-GATA1 S161D S187D were successfully constructed using the high primer PCR method,and expression of the fusion protein was verified. Confocal laser microscopy showed that the fusion protein was mainly located in the nuclei of HEK293 cells. Conclusion A eukaryotic expression vector of a GATA1 mutant was successfully constructed using the large primer method. This work lays the foundation for further studies on the structure and function of the mutant.

Establishment of rat bone mesenchymal stem cells expressing IL-33 / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To explore a way to establish a rat bone mesenchymal stem cell(BMSC)line expressing Interleukin(IL)-33. <p>METHODS: BMSCs were isolated from 6-week-old Wistar Rat. GV303 virus were used as a IL-33 gene carrier to tansfect isolated BMSCs.Three days later, transfected BMSCs were observed under a fluorescence microscope. The expression level of IL-33 of transfected BMSCs was detected by Western blot and ELISA. <p>RESULTS: The BMSCs were successfully isolated, since flow cytometry results showed that rat BMSCs CD90 and CD29 positive, CD45,CD34 and CD11b negtive. Furthermore, BMSCs were able to be differentiated to osteoblasts, adipocytes and chondrocytes respectively. Green fluorescence of GFP BMSCs was observed under the fluorescence microscope 3d after virus transfecting. Expression of IL-33 was detected by Western blot and ELISA in the transfected rat BMSCs. <p>CONCLUSION: A rat BMSC cell line expressing IL-33 was established by GV303 virus transfection.

Establishment and optimization for TALE-TFs construction / 重庆医学

Objective To optimize the method of transcription activator‐like effector transcription factors (TALE‐TFs) con‐struction ,some improvement and adaption were made based on the traditional methods .Methods We first constructed the basic tandem fragments with different length ,including trimer ,tetramer ,pentamer and hexamer by Golden Gate cloning technique and PCR ,then the procedure with the highest efficacy was chosen to construct our TALE‐TFs .To determine the function of the TALE‐TFs ,the plasmid pminCMV with the specific binding sequence of TALE‐TFs was constructed by fragment substitution reaction (FSR) .The transcription activating function of TALE‐TFs was confirmed by the intensity of red fluorescence ,after TALE‐TFs , pEGFP‐N1 and pminCMV plasmid were co‐transfected into 293HEK cells .Results An optimized method for TALE‐TFs construc‐tion and functional assay was established .Conclusion This method can potentially be wildly used in fields that the expression of some constitutively expressed genes needs to be modified .

Constructed a cell line to express hBD1 stablly and detected the antimicrobial activity of hBD1 to multidrug resistant bacterial strains / 中华微生物学和免疫学杂志

ObjectiveTo established a cell line that expresses hBD1 stably,and detected the antimicrobial activity of the hBD1 to the muhidrug resistant bacterial strains.MethodsRecombinant plasmid was introduced into COS-7 cells by lipofectamine,cells were selected in culture medium containing G418 to acquired the monoclonal cell lines,total RNA were extracted from the cultured cells,expression levels of hBD1 mRNA was identified by RT-PCR,collected the supernatant solution of the cultured cell,expression levels of protein was identified by Western blot.Put the expression products and resistant organisms mixed together,after incubation in different times in 37℃,coating the mixtures in LB flat,then obtained the ratios between colonies number of experimental groups and colonies number of control groups,put those ratios as the survival rate of the drug resistance bacterias.Results The monoclonal cell lines had obtained after screened with G418,the hBD1 gene could be detected both at transcriptional and protein levels,Under the influence of expression product hBD1,survival rate of muhidrug-resistant Acinetobacter baumannii,multidrug-resistant Escherichia coli and multidrug-resistant Klebsiella pneumoniae could reduced to 9%,22% and 50%,but survival rate of multidrug-resistant Stenotrophomonas maltophilia is not have apparente difference with the control group.ConclusionThe stably-transfected cell line of hBD1 was successfully constructed,and the expression products of hBD1 showed the antimicrobial activity toward multidrug resistant bacterial strains.

Osteogenic Activity of MSC Infected by Recombinant Adenovirus Vector Ad-LMP-1 / 中山大学学报(医学科学版)

[Objective]This study was designed to construct a recombinant adenovirus vector contains LMP-1 gene,and investigate the osteoinductive activity of MSC which were transfected recombinated adenoviral vector carrying LMP-1 gene.[Methods]Total RNA was extracted from mt osteoblast and the LMP-1 gene was acquired by RT-PCR,the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology,then the entry clone and the expression vector were used to create the expression clone throush the LR recombination reaction.The adenovirus expression clone was linearized by PacI and transfected to the 293A cell line to harvest a high titer.Ad-LMP-1 was infected into the 3rd passage MSC,the expression of LMP-1 was detected by Western blot.The osteogenic activity of MSC was evaluated by the expression of collagen Ⅰ,ALP,osteocalcin and the formation of bone nodule.[Result]The LMP-1 gene was successfully acquired and confirmed,the entry clone and the expression clone were both verified by enzymes digestion,and the expression clone was further confirmed by sequenced.The expression of LMP-1 was detected successfully in MSC.The increasing expression of collagen Ⅰ,osteocalcin.ALP and bone nodule were observed by comparing to the control group.[Conclusion]Gateway technology not only make construction of the pAd-LMP-1 recombination adenovirus vector simple and fast,but also get a high transfection efficacy in MSC.LMP-1 gene can induce the osteoblast differention of MSCs,and improve its osteogenic activity.The adenovirus vector is reliable to be used in further gene therapy research.

Construction of human eukaryotic vector with expression of Survivin in K562 cells / 白血病·淋巴瘤

Objective To construct an eukaryotic vector with expression of human survivin gene with green fluorescent protein which is named pIRES2-EGFP/survivin and transfected into K562 cell line.Methods Using pDNR/Survivin plasmid as a template, the full length of survivin cDNA was amplified by PCR and subsequently cloned into T-A vector and then subcloned into pIRES2-EGFP vector. After identified by digestion of restrictive endonucleases, pIRES2-EGFP/survivin was further confirmed by sequencing. Then it was transfected into K562 cells with superfect reagents. The mRNA was isolated and survivin gene was detected by Western blotting. Results The exact sequences of pIRES2-EGFP/survivin vector were confirmed by digestion of restrictive endonucleases and sequencing. After transfection, the expressions of green fluorescent protein were present. The mRNA expression of survivin has been detected in transfected cells by RT-PCR. Conclusion The vector pIRES2-EGFP/survivin has been constructed and could express survivin gone in K562 cells successfully.

Transfection and Expression of eNOS Gene on Canine Endothelial Cells / 上海第二医科大学学报

Objective To study endothelial nitric oxide synthase (eNOS) gene transfecting endothelial cells (ECs). Methods eNOS gene was transfected into the steady ECs system by cationic liposomal transduction and check the transfection effect by RT-PCR and immunohistochemistry assay. To test the concentrations of NOS, nitric oxide (NO) and von willebrand factor (vWF) in culture media by colorimetry and ELISA, respectively,and transfected EC function was observed. Results The effect of transfection was satisfactory with RT-PCR products electrophoresis, sequence and immunohistochemistry. The concentrations of NOS and NO in transfected EC culture media increased with time (P

Construction and target gene expression of recombinant adenovirus vectors containing vascular endothelial growth factor 165 and angiopoietin-1 / 中国矫形外科杂志

[Objective]To construct the recombinant adenovirus vector co-expressing human vascular endothelial growth factor 165(VEGF165) and angiogenin-1(Ang-1),and to observe the expression of target gene after transfection.[Method]Molecular biologic techniques were used to clone the genes of VEGF165 and Ang-1,and PCR was used to amplify the DNA sequence of IRES(internal ribozyme entry site) from pIRES2-EGFP.The genes of VEGF165,IRES and Ang-1 were subcloned to pTrack-CMV one by one to get the plasmid named pTrack-CMV-VIA,which contained all the three genes.The linearized shuttle plasmid was cotransformed into BJ5183 bacteria with backbone vector AdEasy-1.Further the recombinant plasmid was packaged and amplified in QBI-293A cells after Pac I digestion to get adenovirus vector pAd-VIA.The expression of the gene of interests was evaluated by fluorescence microscopic analysis of GFP expression and enzyme linked immunosorbent assay(ELISA).[Result]The recombinant adenovirus plasmid was consistent with that shown by sequencing and restriction endonucleases digestion.The recombinant pAd-VIA vector was packaged and amplified successfully in QBI-293A cells.The titer was 2?1010 PFU/ml after amplifying.High positive GFP was expressed in the field through fluorescence microscopy after being transfected by recombinant pAd-VIA.ELISA indicated the expression of the target genes.The difference between the tansfected and non-transfected group was significant(P

Study on the toxicity and transfection efficiency of PEI/MYOC gene complexes in vivo / 眼科

Objective To investigate the toxicity and transfection efficiency of PEI-mediated plasmid DNA encoding myocilin (MYOC) gene transfected into trabecular meshwork.Design Experimental study.Participants SD rats.Methods PEI and plasmid encoding MYOC gene complex was prepared and 20?l PEI/DNA complex was directly injected into anterior chamber of SD rat by microinjection. The eyes were extracted at day 1,3,7,and 14 after injection for the structure analysis of trabecular meshwork by HE staining and transmission electron microscope.Expression of the transferred myocilin gene was monitored by fluorescence microscopy. Main Outcome Measures HE staining,immunohistochemistry and electron microscope.Results No significant toxicity or inflammation was detected under the HE staining and electron microscope.PEI/DNA complex were seen in cytoplasm of trabecular meshwork cells. The fluorescence intensity of myocilin expression in the trabecular meshwork cells was increasing with time and peaked on clays 3 and declined afterward.Conclusion Plasmid DNA mediated by PEI could be efficiently transfected into trabecular meshwork cells by directly injecting into anterior chamber without any toxicity.

Inhibitory effect of mouse angiostatin cDNA gene transduction on human liver cancer cell / 中华消化杂志

Objective To study the effect of the mouse angiostatin cDNA gene transfecting into human liver cancer cell line HCC7721 on cell growth, cell cycle phase distribution, cell morphology in vitro and tumorigenesis in vivo , and the mechanisms. Methods The gene fragment of mouse angiostatin cDNA was directly cloned into an eukaryotic expression plasmid pcDNA3.1(+) of the promoter CMV between the multicloning sites HindⅢ and XbaⅠ and confirmed the correct recombinant plasmid pcDNA3.1(+) angio through enzymatic digestion and gene sequencing. Then it was transfected into human liver cancer cell line HCC7721 with liposome, pcDNA3.1(+) as vecter control and liposome as mock control. After 30 day selection by neomycin G418, angiostatin expression at the levels of mRNA and protein in vitro were tested by RNA dot blot and FACS respectively. Cell morphology under was observed light microscope, made growth curves were made, and cell cycle distribution checked in FACS. Animal model was set up to study tumorigenesis in vivo of those cells transplanted subcutaneously in the right hind legs of nude mice BALBc. Microvessel density (MVD) was analyzed and angiostatin expression in the tumor tissues by in situ immunohistochemistry. Results The recombinant eukaryotic expression plasmid pcDNA3.1(+) angio was constructed Auccessfully. After selection of G418 for about 4 weeks there were macroscopic cell clones in the experimental group and vector control group, but no survival cells in the mock control. In vitro angiostatin were expressed in the experimental group, but not in the vector control. There weren't significant changes in cell morphology, cell growth curves and cell cycle distribution between the experimental and the control group. However, the nude mice experiment showed that tumorigenic capability of the experimental cells had been reduced greatly. Immunochemistry study showed that there were much less microvessels and conversely stronger angiostatin positive staining in the tumor tissues than in the control group. Conclusions Mouse angiostatin has no direct inhibition on human liver cancer cell line HCC7721 in vitro , but it inhibits effectively the tumorigenesis of HCC7721 in vivo , which is probably due to its inhibition on tumor angiogenesis in a paracrine path way.

Establishment of a cell line of Tca8113 with stable expression of nm23-H1 by gene transfection / 实用口腔医学杂志

Objective:To establish a tongue cancer cell line that stably overexpresses nm23-H1.Methods:The reconstructed plasmid, pCMV-BamH1-Neo-nm23-H1, was transfected into bacteria and amplified. The plasmid was identified by electrophoresis on a 10 g/L agarose gel and stained with ethidium bromide and then transfected to the cell line of Tca8113 by lipofectin strategy and was selected by G418 for 5 weeks. The stable expression of nm23-H1 was identified by immunofluorescence,Western-blotting and flow cytometer.Results:Restriction endonuclease Bam H1 examination showed that 986 bp of nm23-H1 was in pCMV-Bam-Neo vector. 5 weeks after transfection positive clones were obtained and the transfected cells were proliferated to confluent.Immunohistochemical examination,Western blot and flow cytometry showed that nm23-H1 expression was increased in the transfected cells.Conclusion:A tongue cancer cell line expressing nm23-H1 is established.

Investigation of the inhibition of the cell growth and down-regulation of mTOR in the cholangiocarcinoma QBC939 cells transfected with plasmid PTEN in vitro / 中国普通外科杂志

Objective To investigate the effects of the tumor suppressor gene PTEN in growing inhibition and down-regulating mTOR in cholangiocarcinoma QBC939 cells in vitro.Methods QBC939 cells were transfected with plasmids wild-type PTEN and C124S-PTEN in vitro.After transfection,the expression of the PTEN and phosphorylation of AKT and mTOR was detected by Western blot.Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells.Results Compared with the control,the expression of phosphorylation AKT was decreased and mTOR were down-regulated respectively when transfected with the wild-type PTEN.However,after transfection with mutation-type PTEN,the level of PTEN in the cells by increased,but phosphorylation AKT level and mTOR expression had no significant change.Conclusions PTEN can be actived by phosphorylated AKT.Actived AKT decreased the mTOR which led to tumor cells apoptosis and regulation of the tumor cell cycle.In the pathway of signal transmission of PI3K/AKT/PTEN/mTOR,PTEN and mTOR are closely related through phosphorylation of AKT.

Cloning of human REG ? cDNA and construction of the stable transfective cell line / 重庆医科大学学报

Objective:To construct the stable transfective cell line with the eukaryotic expression vector for human REG?cDNA to laid the foundation for further study of the function of REG?.Methods:REG?cDNA was obtained by RT-PCR of total RNA extracted from the human breast cancer cell MCF-7,and it was digested by the restriction endonuclease EcoRⅠand EcoRⅤbefore connection withPcDNA3.1.The recombination with forward insert were slelected by restriction endonuclease digestion and confirmed correct by sequencing and then transfected into HBL-100 cell by using lipofectamine2000.The stable transfected cell line was selected in medium containing antibiotic(G418).Results:The result of immunocytochemistry and RT-PCR of total RNAextracted from the stable transfected cell line showed that there existed the overexpression of REG?cDNA in it.Conclusion:The construction of the eukaryotic expression vector for REG?was successful,and the stable transfected cell line for overexpression REG? was obtained by G418 selection.This work may pave the way for further study of the functions of REG?.

Construction of pIRES-I-A~d?? and stable expression of BALB/c mouse I-A~d?? chain genes in NIH3T3 cell line / 中国免疫学杂志

Objective:To construct pIRES-I-Ad?? bicistronic eukaryotic expression vector. Methods: Total RNA was acquired by TRIZOL method, the genes of I-Ad ?? chains were amplified through RT-PCR, respectively. The target genes were connected to pGEM-T vector and sequenced. Then the target genes were subcloned into pIRES bicistronic eukaiyotic expression vector and NIH3T3 cell line was transfected. Transfectant was screened by G418 antibiotics. Total RNA of transfectant was obtained by TRIZOL method, mRNA of foreign gene was examined by RT-PCR. Flow cytometry( FCM) was used to detennine foreign gene expression in protein level and surface expression in NIH3T3 cell line. Results: Bicistronic eukaryotic expression vector pIRES-I-AdaB was established. Foreign gene in mRNA level in transfectants was examined. Verified Ⅰ-Ad ?? chain wa3 expressed in high level expressed on transfected NIH3T3 cell line surface by FCM. Conclusion:Bicistronic eukaryotic expression vector pIRES-I-Ad ?? was constructed successfully. It is useful for studying antigen presentation and interaction between epitopes and MHC- Ⅱ molecule of BALB/c mouse.

THE INFLUENTIAL FACTORS AND OPTIMUM CONDITIONS OF ELECTRIC TRANSFECTION ON DENDRITIC CELLS / 解剖学报

Objective To investigate the method and optimum conditions of electric transfection,and the major influential factors of electransfection efficiency and the survival rate of dendritic cells. Methods RNA was extracted from human hepatocarcinoma cell line(Bel 7402).Purified monocytes as precursor DC-s(pDC-s) were separated from human peripheral blood cells(PBMCs) by density gradient centrifugalization with lymphocyte gradation fluid and adherence method,pDCs were incubated in RPMI-1640 medium containing rhGM-CSF(8?10~5IU/L) and rIL-4(5?10~5IU/L) for 7 days and made them fully differentiate into immature DCs(imDCs).The total RNA human hepatocarcinoma cell and green fluorescent protein(GFP) were electransfected respectively into imDCs by electroporation apparatus with different electric voltages,times of impulse,cell concentrations,temperatures and electroporation buffers.Numbers of green fluorescence positive cells and the total cell number were counted respectively under fluorescent microscope,and visible light microscope.One day after the electric transfection,the cells were stained with 0.4% trypan blue,and electransfection efficiency and the cell survival rate were counted. Results Electransfection efficiency was increased to the highest value,up to about 49.7% when imDCs with the concentration of 5?10~6 cells/ml were mixed with 40?g-total RNA of human hepatocarcinoma cell,the electric voltage of electroporation apparatus was set at 300V,and the time of impulse was 500Us.Conclusion Electric transfection provides a technical possibility to make human hepatocarcinoma RNA into imDCs.The major influential factors of the electransfection efficiency were electric voltage and impulsing time.As receptor cells,the imDCs growing condition was also an important influential factor.