ABSTRACT
BACKGROUND: In the research of human embryonic stem cells, introducing exogenous molecules such as DNA into cells is a common research method, but the transfection efficiency is relatively low. It is crucial to answer the question of how to optimize the existing conditions to improve the transfection efficiency. OBJECTIVE: To compare the effects of two different passaging methods on H9 transfection efficiency, in order to optimize the conditions required for embryonic stem cell transfection. METHODS: Human embryonic stem cell lines H9 were cultured for 48 hours after small clone passaging or single-cell passaging. Lipofectamine 3000 was used to transfect pAdTrack-AKT1 fluorescent plasmid into human embryonic stem cells. After 2 days of transfection, the expression of fluorescent plasmids was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry. RT-qPCR and western blot were used to detect the mRNA and protein expression levels of AKT1 respectively. RESULTS AND CONCLUSION: Under the fluorescence microscopy, the number of cells expressing fluorescent plasmids in the single-cell passaging group was more than that in the small clone passaging group, and the flow cytometry analysis showed that the transfection efficiency of cells in the single-cell passaging group was (47.18±2.00)%, which was significantly higher than (19.52±0.86)% in the small clone passaging group (P < 0.01). RT-qPCR and western blot analysis showed that the expression levels of AKT1 mRNA and protein in the single-cell passaging group were significantly higher than those in the small clone passaging group (P < 0.01). These findings indicate that single-cell passaging can increase the contact area between cells and transfection reagent liposomes, and improve the transfection efficiency of human embryonic stem cells.
ABSTRACT
Objective@#To observe the changes of PD-1 expression, mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells.@*Methods@#The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells. The expression of PD-1 was analyzed by flow cytometry. The mRNA level of PD-1 was analyzed by PCR. The cell proliferation was analyzed by CCK-8 assay. The cytotoxicity was analyzed by LDH assay.@*Results@#①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same (P>0.05) . ②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells, with or without PD-1 inhibitor were as the same (P>0.05) . ③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells (P<0.001) . There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer (P>0.05) . ④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process (P>0.05) . There was no difference of mRNA level of PD-1 in all groups during the culture process (P>0.05) . ⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells (P<0.001) . The PD-1 inhibitors could antagonize this effect. There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells (P>0.05) .@*Conclusion@#The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells. However, the mRNA level of PD-1 of all groups did not change, even if PD-1 inhibitor was applied.
ABSTRACT
Objective To investigate the factors affecting the success rate and cell survival rate of electroporation-mediated transfection of human primary fibroblasts by optimizing the electroporation parameters, so as to screen the optimal conditions for electroporation-mediated transfection of human primary fibroblasts. Methods Plasmid pEGFP-N1 containing enhanced green fluorescent protein (EGFP) gene was transfected into human primary fibroblasts with electroporation by changing the conditions such as pulse voltage, pulse number and plasmid concentration. Cell mortality rate was detected by trypan blue staining. Transfection efficiency was measured by flow cytometry at 24 h after transfection. Results The electroporation-mediated transfection effect of human primary fibroblasts was optimal under the following condition: low voltage mode, pulse voltage of 320 V, plasmid concentration of 20 pg/mL and single pulse, with the cell transfection efficiency being (42.53 ± 0.63)% and the cell survival rate being (74.30 ± 3.01)%. Conclusion The electroporation-mediated transfection efficiency of primary human fibroblasts can be improved by optimizing the transfection conditions, which might lay a foundation for subsequent function research of gene.
ABSTRACT
ABSTRACT Cationic polymers such as polyallylamine (PAA) having primary amino groups are poor transfection agents and possess a high cytotoxicity index when used without any chemical modification. In this study, PAA was modified with cholesterol in order to improve transfection efficiency and to reduce cytotoxicity. PAA polymers with molecular weights of 15 and 65 kDa were selected and grafted with cholesterol at percentages of 5, 10, 15, 30, and 50. After purification, the efficacy of the synthetic vectors was evaluated in terms of DNA condensation using the ethidium bromide test, buffering capacity, particle size, zeta potential, transfection efficiency, and cytotoxicity assay in Neuro2A cell lines. According to the ethidium bromide test, these vectors can condense DNA at moderate and high carrier to plasmid (C/P) ratios. The buffering capacity of the prepared vector in both molecular weights was less than unmodified PAA. Particle size measurements demonstrated that modified PAAs were able to form nanoparticles ranging in size from 125 to 530 nm. The vectors based on PAA 15 kDa demonstrated a better transfection efficiency than the vectors made of PAA 65 kDa. Cytotoxicity studies showed that toxicity of all vectors was less than PAA. Some cholesterol modified polymers composed of PAA (15 kDa) were suitable vectors for gene delivery with low cytotoxicity.
Subject(s)
Genetic Therapy , Cholesterol/therapeutic use , Polymers , Transfection/instrumentationABSTRACT
Objective To prepare PCM modified liposome (PCM-LIP) containing enhanced green fluorescent protein expression plasmid (pEGFP) and to evaluate its myocardial targeting ability. Methods Liposome was prepared by film-ultrasonic, with PCM used as ligand and DOTAP as cationic lipid material. PCM-LIP containing pEGFP was prepared by incubating liposome with pEGFP at room temperature. The connecting method of PCM was optimized and the connection rate of PCM was determined. The characteristics of liposomes including encapsulation ability, morphology, particle size, zeta potential and stability in phosphate buffer solution (PBS) were observed. The transfection efficiency of liposomes into H9c2 cells was evaluated by inverted fluorescence microscopy and flow cytometry, so as to characterize their myocardial targeting ability and to determine the optimum dosage of PCM. Results PCM-LIP was prepared by insertion method and the amount of PCM accounted for 3% of the lipid. After incubation with pEGFP, PCM-LIP was spherical in shape, with the particle size being (261. 9±2. 2) nm, zeta potential being (-5. 0±0. 6) mV, and PCM-LIP was stable in PBS at -4℃ for 30 d. The transfection efficiency of PCM-LIP was significantly higher than that of unmodified liposome (P<0. 05). Conclusion PCM can improve the transfection efficiency of liposome into cardiomyocytes and PCM-LIP shows a satisfactory myocardial targeting ability.
ABSTRACT
Objective To investigate the efficiency of target gene transfection of the heart and liver after tail vein or intramyocardial injection of adenovirus vector (GFP-Ad).Methods GFP-AD was constructed at first.A total of 20 male 8-week old C57BL/6 mice were randomly and equally divided into tail vein injection of GFP-AD group and intramyocardial injection of GFP-AD group.The mRNA levels of GFP in the heart and liver tissues were detected by Q-PCR at different time points.Fluorescence microscopy was performed to visualize the expression of GFP fluorescence.Results Compared with the tail vein injection group, the GFP mRNA level in mouse heart tissue was apparently higher in the intramyocardial injection group.In both groups, the GFP mRNA levels in liver tissue were significantly increased compared with that in the heart tissue.In the tail vein injection group, the GFP mRNA level in liver tissue reached a peak on day 7;but in the intramyocardial injection group, the mRNA level of GFP in liver tissue reached apeak on day 3.We also observed the same trend of GFP fluorescence expression in the tail vein injection group compared with that in the intramyocardial injection group.Conclusions Intramyocardial injection of adenovirus vector is suitable to achieve a higher transfection efficiency in mouse heart tissue compared with the tail vein injection method.Although both injection methods are suitable for transfection of mouse liver, the tail vein injection method is preferential for it is simple and less invasive.
ABSTRACT
Objective To compare the transfection efficiency of cationic polyethylenimine(PEI)with Lipofectamine 2000TM by using the plasmid DNA encoding vascular endothelial cell growth factor (VEGF165 )gene in human embryonic kidney cell line 293T.Methods PEI of different N/P ratio and Lipofectamine 2000TM were used to deliver the vector containing VEGF165 to 293T cells,respectively.Green fluorescent protein(GFP)gene was inserted into the vector as a report gene.Evaluation of cytoactive was performed by CCK-8 assay 24 h after transfection.The cells were observed by fluorescent microscope and the presence of VEGF165 in cell supernatant was detected by ELISA 48 h after transfection.The transfection efficiency was calculated and com-pared.Results Similar cytoactive and best transfection efficiency could be obtained when N/P ratio was 9,the transfection efficien-cy was around 70%.Furthermore,the presence of VEGF165 increased significantly after transfection(P <0.05),but there was no significant difference between the two groups in which different transfection methods were adopted.Conclusion PEI as a novel oli-gofectamine reagent could mediate more efficient transfection compared with lipofectamine.It also has low cell-toxicity and low price and could be an ideal vector in gene delivery technology.
ABSTRACT
Background Follistatin (FST), a secreted glycoprotein, is intrinsically linked to muscle hypertrophy. To explore the function of duck FST in myoblast proliferation and differentiation, the pEGFP-FST eukaryotic expression vector was constructed and identified. The biological activities of this vector were analyzed by transfecting pEGFP-FST into cultured duck myoblasts using Lipofectamine 2000 and subsequently determining the mRNA expression profiles of FST and myostatin (MSTN). Results The duck pEGFP-FST vector was successfully constructed and was confirmed to have high liposome-mediated transfection efficiency in duck myoblasts. Additionally, myoblasts transfected with pEGFP-FST had a higher biological activity. Significantly, the overexpression of FST in these cells significantly inhibited the mRNA expression of MSTN (a target gene that is negatively regulated by FST). Conclusions The duck pEGFP-FST vector has been constructed successfully and exhibits biological activity by promoting myoblast proliferation and differentiation in vitro.
Subject(s)
Animals , Transfection , Myoblasts/metabolism , Follistatin/metabolism , Hypertrophy , Muscular Diseases/pathology , Biological Assay , In Vitro Techniques , RNA, Messenger , Cell Differentiation , Cell Proliferation , Ducks , Eukaryotic Cells/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
Objective To prepare the ternary nano-gene polyplex by adding hyaluronic acid (HA) into DNA/polyamidoamine (PAMAM) binary nano-gene polyplex, and to probe into its gene transfection ability. Methods DNA and HA were mixed with PAMAM dendrimer in different charge ratios to form ternary nano-gene polyplex, and the size and zeta potential of the polyplex were measured. MCF-7 and MDA-MB-231 cells were used for transfection efficiency test; B16 and MCF-7 cells were used for cell viability assay. Results and conclusion The prepared ternary nano-gene polyplex has even size and stable structure. HA can enhance the gene transfection efficiency and reduce the cell toxicity compared with pure PAMAM, making it suitable for gene delivery and transfection.
ABSTRACT
Objective: To synthesize a new type of cationic polymers (β-ammo esters) (PBAE) and to study its role as a gene vector. Methods: PBAE was synthesized by the Michael addition reaction. PBAE/pDNA complex nanoparticles were prepared by self-assembly, and the gene tranfection efficiency mediated by the nanoparticles into HEK293 cells in vitro was observed. Results: When weight ratio of PBAE/pDNA was 50: 1, pDNA was completely wrapped. And the transfection efficiency of PBAE/pDNA nanoparticles (PBAE/pDNA weight ratio being 200: 1) was significantly higher than PEI/pDNA nanoparticles (43.3% ± 3.7% vs 30.3% ± 2.1%, P < 00.05). Conclution: Cationic poly(β-amino esters) can electrostatically bind to and condense anionic DNA into nanometersized parties. PBAE/pDNA nanopartides has a high transfection efficiency in in vitro transfection experiments.
ABSTRACT
The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20 percent O2) and hypoxia (less than 5 percent O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50 percent lower under hypoxia, while the HRE plasmid was about 50 percent higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.
Subject(s)
Animals , Humans , Male , Mice , Base Sequence/genetics , Cell Hypoxia/genetics , Gene Expression/genetics , /genetics , Vascular Endothelial Growth Factor A/metabolism , Cell Line , Enzyme-Linked Immunosorbent Assay , Gene Targeting , Genetic Vectors/genetics , Mice, Inbred BALB C , Polymerase Chain Reaction , Plasmids/genetics , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Aim: To study the effects of molecule weight,the N/P ratio,solvent and ionic strength on the formation and surface properties of PEI/DNA complexes,and study its transfection efficiency.Methods: The N/P ratio of the prepared PEI/DNA complexes was optimized using gel electrophoresis and UV quantitative assay.The particle size and surface charge of the complexes were measured in different solvents and ionic strength.The transfection effi-ciency in HepG2 cells was observed.Results: There was a positive correlation between combination of PEI and DNA and molecule weight of PEI.In addition,the surface properties of PEI/DNA complexes were also influenced by the solvents and ionic strength.Comparable transfection efficiencies in HepG2 cells were observed for the Lipo-fectamine 2000/DNA and the prepared PEI(25 kD)/DNA complexes in PBS at the N/P ratio of 12-15,which was much higher than that of naked DNA.Conclusion: The optimized PEI/DNA complexes could effeciently transfect the cells in comparison to the positive control.
ABSTRACT
BACKGROUND: Small interfering RNAs (siRNAs) have been used to knockdown specific gene expression in various cells. Astrocytes and microglial cells play a key role in fundamental central nervous system functions and in chronic neuroinflammation. The aims of this study were to determine the optimal concentration of siRNA demonstrating efficient transfection and inhibition of gene expression via RNA interference (RNAi) and lower cytotoxicity, in primary cultured astrocytes and microglial cells of rats. METHODS: Astrocytes and microglial cells were isolated from the cerebral cortices of 2-day-old rats. Both the cells were transfected using transfection reagent (Lipofectaminetrade mark 2000), and fluorescein-labeled double-stranded RNA (dsRNA) or siRNA targeting green fluorescent protein. Transfection efficiency and cytotoxicity of dsRNA, and the degrees of RNAi induced by siRNA in these cells, were evaluated at various concentrations of RNA. RESULTS: Transfection efficiencies of dsRNA in both astrocytes and microglial cells were significantly higher (P < 0.05) at the concentrations of 20, 40, and 80 nM than at the concentrations of 0, 5, and 10 nM. There were no significant cytotoxicities within the applied concentrations of dsRNA (0-80 nM). The degrees of RNAi induced by siRNA were significantly higher (P < 0.05) at the concentrations of 5, 10, 20, 40, 80 nM, and 20, 40, 80 nM in astrocytes and microglial cells, respectively, compared with the control (0 nM). CONCLUSIONS: The siRNA concentration of 20 nM may be appropriate to induce RNAi in both astrocytes and microglial cells, while demonstrating low cytotoxicity, high transfection efficiency, and effective RNAi.
Subject(s)
Animals , Rats , Astrocytes , Central Nervous System , Cerebral Cortex , Gene Expression , Gene Silencing , RNA Interference , RNA, Double-Stranded , RNA, Small Interfering , TransfectionABSTRACT
Objective:To enhance the transfection efficiency and to reduce the toxicity of the polyethyleneimine(PEI),we synthesized PEI derivatives and tested their toxicity and transfection efficiency in different cell lines. Methods:We first developed PEGylated PEI to decrease the toxicity of PEI,and then we conjugated folate on the distal end of the novel PEG-PEI to introduce specificity for special tumor cells.Following we checked the characterization of polymers and tested their toxicity and transfection efficiency in three cell lines with MTT assay,EGFP/fluorescent image,reporter assay and flow cytometry. Results:These copolymers effectively condensed plasmid DNA(pDNA) into nanoparticles with positive surface charge under a sui table N/P ratio.These derivatives reduced the cytotoxicity of PEI 25ku in different cell lines(i.e.,HEK 293T,glioma C6 and hepatoma HepG2 cells).More importantly,compared with PEI 25ku,the transfection efficiency was increased. Reporter assay and flow cytometry showed that FA-PEG-PEI/pDNA complexes exhibited higher transgene activity than that of PEG-PEI/pDNA or PEI/pDNA in folate-receptor(FR) positive(HEK 293T and C6) cells but not FR-negative(HepG2) cells. Conclusion:These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristic of good biocompatibility and relatively high gene transfection efficiency.
ABSTRACT
Cyclodextrin(CD) is gradually applied in the nonviral gene vector system,due to its biocompatibility and flexibility of tailing via structural modification,polymerization or supramolecular combination.The ideas and research progress of the CD,its low molecular derivatives,CD polymers and CD supramolecular combination in the field of norviral gene vectros were reviewed,and their "structure-safety-transfection efficiency" relationships were discussed.
ABSTRACT
Objective:To establish high efficient transfection and expression system in vitro for phPPAR?1-IRES2-EGFP re- combinant plasmid carrying human peroxisome proliferator-activated receptor?1(hPPAR?1)gene.Methods:Cationic lipid trans- fection reagent lipofectamine 2000 was used to transfect phPPAR?1-IRES2-EGFP recombinant plasmid and control pIRES2- EGFP plasmid to 293 cells.The profiles of GFP expression and transfection efficiency were measured by fluorescence mi- croscopy.Expression of PPAR?1 in transfected 293 cells were determined by werstern blot and real time quantitative poly- merase chain reaction.Results:GFP expression level was high in transfeced 293 cells,the transfection efficiency of phP- PAR?1-IRES2-EGFP was(83?11)%.In transfected 293 cells,high efficient expression of hPPAR?1 gene were detected both at mRNA and protein levels by real-time PCR and western blot analyses.Conclusion:High efficient transfection and expres- sion system for phPPAR?1-IRES2-EGFP recombinant plasmid in vitro is successfully established,which provide a useful tool in investigating hPPAR?gene function as well as establishing molecular platform by which the candidates of unknown hP- PAR?ligands or activators can be found.
ABSTRACT
Objective To investigate the function of transferrin-DNA complex,transported by transferrin(Tf)and trans-arterial injection via interventional approach be the duel-target-orientated delivery and the transferring into malignant cells to get more effective therapy.Methods p53-LipofectAMINE ligand with different concentrations of Tf(0,10,25,50,100?g)transfected the 4 strains including LM6、Hep3B、YY and L02 in vitro to evaluate the gene transfeetion efficiency through western blot.Then,after setting up the VX2 hepatocarcinoma models,we delivered the Tf-p53-LipofeetAMINE complex into the hepatic arteries via interventional techniques to analyse the transfection efficiency in vivo.Results Tf,within the range of 10 100?g,could increase gene transfection efficiency mediated by liposome,and the efficiency increases with the raise of Tf concentration.Combination with interventional technique to inject Tf-DNA complex into tumor arteries,gene transfeetion efficiency was enhanced in rabbit models.Conclusion Tf can enhance gene- liposome transfection efficiency,furthermore with combination of interventional catheter technique,there would be a potential duel-target-orientated gene therapy method.(J Intervent Radiol,2007,16:99-103)
ABSTRACT
Objective To investigate the transfection and expression of p53 genes mediated by liposome and its feasibility in treatment of liver cancer by transcatheter arterial injection on rabbit VX2 hepatocarcinoma model.Methods pCMV-myc-p53 plasmids,LipofectAMINE and p53-LipofectAMINE complex were infused into tumor's feeding artery of rabbit VX2 hepatocarcinoma model,respectively,and then protein of cancer tissue was extracted,followed by measuring gene transfection and expression by western blot and immunohistochemistry,p53-LipofectAMINE complex in different doses were infused into tumor's feeding artery of rabbit VX2 hepatocarcinoma model with the gene transfection and expression detected by the same way.Results Liposome-mediated p53 gene injected through catheter could be successfully transfected and expressed in the cancer tissue of rabbit VX2 hepatocarcinoma model,with transfection efficiency higher than the gene delivery alone.The efficiency and the gene dose has dose-effect relationship.Condusions Treatment of liver cancer by transcatheter arterial injection of p53 genes mediated by liposome is a feasible and effective method,with wide prospect of application.(J Intervent Radiol,2007,16:109-114)