In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6 μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6. 0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4 μg/well with PEI/plasmid ratio of 3 : 1-5 : 1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.
Green Fluorescent Proteins ; HEK293 Cells ; Humans ; Plasmids ; Polyethyleneimine ; Transfection ; methods

Apoptosis is a physiologically essential mechanism of cell and plays an important role in reducing the development and progression of tumors. The appealing strategy for cancer therapy is to target the lesions that induce apoptosis in cancer cells. Survivin, the smallest member of the mammalian inhibitors of the apoptosis protein family, is upregulated in various malignancies to protect cells from apoptosis. Survivin knockdown could induce cancer cell apoptosis and inhibit tumor-angiogenesis. Survivin expression would be silenced by microRNA (miRNA)-mediated RNA interference. However, noninvasive and tissue-specific gene delivery techniques remain absent recently and the utilizations of miRNA expression vectors have been limited by inefficient delivery technique, especially in vivo. On the other hand, safe and promising technologies of gene transfection would be valuable in clinical gene therapy. Successful treatment of gene transfer method would lead to a new and readily available approach in the anticancer research. Sonoporation is an alternative technique of gene delivery that uses ultrasound targeted microbubble destruction to create pores in the cell membrane. Based on our previous studies, in this article, we postulated that the transfection of miRNA could be mediated by the combination of sonoporation and polyethylenimine (PEI) which was one of the most effective poly-cationic gene vectors and enhance the endocytosis of plasmids DNA and hypothesized that the gene silencing and apoptosis induction with miRNA targeting human Survivin would be improved by this novel technique. In our opinion, this novel combination of sonoporation and PEI could enhance targeted gene delivery effectively and might be a feasible, novel candidate for gene therapy.
Genetic Therapy ; methods ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; MicroRNAs ; genetics ; Neoplasms ; therapy ; Polyethyleneimine ; chemistry ; Transfection ; methods

Polyethylenimine (PEI) is a kind of nanometer nonviral vector frequently applied in gene transfection. It is simple and easy to prepare and to modify and relatively safe compared to viral vectors. In recent years, PEI has been utilized in many research areas for gene delivery to stem cells in vitro or targeted gene delivery to cells in the brain. This review reveals that the cytotoxicity and low transfection efficiency of PEI requires to be improved. However brain-targeted modification indicates the promising prospect of PEI for gene therapy in cerebrovascular diseases.
Genetic Therapy ; Genetic Vectors ; Humans ; Nanostructures ; chemistry ; Polyethyleneimine ; chemistry ; Stem Cell Transplantation ; methods ; Transfection ; methods

The genome instability and tumorigenicity of induced pluripotent stem cells (iPSC) hinder their great potentials for clinical application. Using episomal vectors to generate iPSC is the best way to solve safety issues at present. This method is simple and the exogenous gene was not integrated into the host genome. However, the reprogramming efficiency for this method is very low and thus limits its usage. This study was purposed to improve episomal method for generating induced pluripotent stem cells from cord blood mononuclear cells (CB MNC), to establish integration-free iPSC technology system, and to lay the foundation for individualized iPSC for future clinical uses. To improve the reprogramming efficiency for iPSC, episomal method was used at various combinations of episomal vectors, pre-stimulating culture mediums and oxygen condition were tested to optimize the method. The results showed that using erythroid culture medium for culturing 8 days, transfecting with episomal vectors with SFFV (spleen focus forming virus) promoter under the hypoxic condition (3%), CB MNC could be mostly efficiently reprogrammed with the efficiency 0.12%. Furthermore, the results showed that erythroblasts (CD36(+)CD71(+)CD235a(low)) were the cells that are reprogrammed with high efficiency after culture for 8 days. It is concluded that a highly efficient and safe method for generation of integration-free iPSC is successfully established, which is useable in clinical study.
Cell Culture Techniques ; methods ; Cellular Reprogramming ; Genetic Vectors ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Plasmids ; Transfection

To explore feasibility of real-time quantitative PCR in assessing efficiency of gene transfection, clonal PCR was employed to analyze efficiency of retroviral-mediated neo gene transfection in primary myoblast, simultaneous real-time PCR were performed for estimation of transfection efficiency; for measuring integrated gene copy number per cell, linear amplification mediated-PCR (LAM-PCR) and retroviral 5'LTR integration analysis also were used. The results showed that: (1) the data from clonal PCR are similar as that from real-time PCR in low efficiency of transfection (< 36%); but in high efficiency of transfection, it is significantly differentiation between clonal PCR and real-time PCR. (2) One copy of transduced gene per cell was observed in retroviral-mediated gene transfection in primary myoblast. It is concluded that real-time PCR can be used to estimate gene transfer vector in vivo, but it is not available for assessing gene transfection in vitro, because high efficiency of transfection could be obtained in most of gene transfection in vitro.
Animals ; Genetic Vectors ; genetics ; Polymerase Chain Reaction ; methods ; Retroviridae ; genetics ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo develop a RP-HPLC method for the determination of quercetin in UGT1A3 cDNA-transfected cells.

METHODSThe lysate of cells transfected with human recombinant uridine 5-diphosphate glucuronosyltransferases UGT1A3 cDNA was co-incubated with quercetin, the reaction was terminated with acetonitrile, and luteolin was used as internal standard. The determination was performed on a C(1) reversed phase column with a mobile phase of methanol-0.1% formic acid (V/V) at a flow rate of 1.0 ml/min. The gradient elution was as follows: 0 - 25 min (30:70-80:20, methanol:0.1% formic acid), > 25-25.5 min (80:20), >25.5-27 min (80:20-30:70), > 27-30 min (30:70). A UV-VIS detector was operated at 368 nm.

RESULTThe standard curve was linear over the concentration range of 5-200 μmol/L (r = 0.9999). The limit of detection was 1.25 μmol/L(S/N ≥ 3), and the limit of quantification was 5 μmol/L (S/N >10, RSD = 6.99%). The method afforded recoveries of 99.1%-103.5%, and precisions for inter- and intra-assay were < 2.5% and < 8%, respectively. In addition, kinetic analysis indicated that the K(m), V(max) and CL(int) (V(max)/K(m)) values for quercetin glucuronide were (62.95 ± 13.16) μ mol/L, (284.50 ± 24.35)nmol*min⁻¹*g⁻¹ and 4.52 ml*min⁻¹*g⁻¹, respectively.

CONCLUSIONThe method established is accurate and simple and suitable for the determination of quercetin in UGT1A3 cDNA-expressed cells.

Cells, Cultured ; Chromatography, High Pressure Liquid ; methods ; Glucuronosyltransferase ; genetics ; Humans ; Quercetin ; analysis ; pharmacokinetics ; Transfection

Carcinoma, Hepatocellular ; therapy ; Genetic Therapy ; methods ; Humans ; Liver Neoplasms ; therapy ; Transfection

We developed a detailed electroporation method to deliver efficiently siRNA into mouse preimplantation embryos. By introducing Cy3 labeled negative control small interfering RNA (siRNA) into mouse preimplantation embryos, we optimized conditions for the electroporation, including the voltage, pulse duration, pulse number, electroporation buffer and an important step to weaken the zona pellucida. Embryonic survival rate, transfection rate and blastocyst development rate were evaluated under the converted fluorescence microscope, by embryos counting and statistical analysis. The best transfection was achieved in opti-MEM under the conditions of 30 V, 1 ms, 3 pulses, and the duration of digestion in tyrode's solution was 10 s. In conclusion, the proposed electroporation approach here is a simple and efficient tool to deliver siRNA for RNA interference (RNAi) into mouse preimplantation embryos.
Animals ; Blastocyst ; metabolism ; Electroporation ; Female ; Male ; Mice ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; methods

OBJECTIVETo investigate the effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit.

METHODSBilateral mandibular osteotomy was performed in 45 New-Zeland rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0.8 mm/day for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups. 2 microg (0.1 microg/microl) of pIRES-hVEGF165-hBMP2, recombinant plasmid pIRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES and the same volume of normal saline (NS) was injected into the distraction area in each group, respectively. After injection, electroporation was performed in every group. Three animals in each group were sacrificed at 7, 14, and 28 days after completion of distraction, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations. The expression of cyclins A, D1 ,E in positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with the single factor analysis of variance and q test.

RESULTSCyclins A, D1, E staining was mainly located in inflammatory cells, granulation tissue monocyte, fibroblast, osteoblasts, osteocyte and the connective tissues around the new bone. The expression reached to the peak at 7th day of consolidation, and decreased at 14th day, and weak at 28th day. Image analysis results showed that, at 7th day, the expression absorbance A in group C (0.59 +/- 0.14) was the strongest, compared to group A (0.41 +/- 0.13), B (0.38 +/- 0.14), D (0.34 +/- 0.12) and E (0.31 +/- 0.10), showing a significant difference (P < 0.05, P < 0.01). There was no significance difference between group A and B (P > 0.05), but the difference between group A/B and group D/E (P < 0.05). At 14th and 28th day, there was no significant difference among group A (0.39 +/- 0.11), B (0.34 +/- 0.10) and C (0.33 +/- 0.09) (P > 0.05), but there was significant difference between group A/B/C and group D (0.19 +/- 0.12) or E (0.14 +/- 0.04) (P < 0.05 or P < 0.01).

CONCLUSIONSElectroporation-mediated gene transfection can promote cyclins A, D1, E expression effectively, which may promote cell differentiation and proliferation, stimulate extracellular matrix synthesis and new bone formation in distraction gap.

Animals ; Cyclins ; metabolism ; Electroporation ; Genetic Therapy ; Mandible ; surgery ; Osteogenesis, Distraction ; methods ; Plasmids ; Rabbits ; Transfection

OBJECTIVETo compare three different methods in transfection of plasmid pGPU6/GFP/Neo into chicken embryo fibroblast.

METHODSDifferent doses (1.25 μl,2 μl,2.5 μl) of Lipofec-tamin2000, Gbfectene-Elite and HilyMax were used to transfect 1 μg plasmid pGPU6/GFP/Neo. The transfection efficiency was observed by the fluorescence and the cell viabilities were measured.

RESULTSThe transfection efficiency of HilyMax was significant higher than that of Lipofectamin2000 and Gbfectene-Elite (86.85%±2.32% compared with 48.33%±3.24% and compared with 37.35%±5.41%; F=18.882, P<0.05). The transfection efficiency of 2.5 μl HilyMax was highest(90.53%±1.15%). The cell viability of Lipofectamin2000 was significantly lower than that of HilyMax and Gbfectene-Elite(65.76%±5.78% compared with 89.54%±0.86% and compared with 82.45%±3.56%;F=90.676, P<0.05).

CONCLUSIONHilyMax has the highest transfection efficiency and the lowest cell toxicity in transfection of plasmid pGPU6/GFP/Neo into chicken embryo fibroblast, with the optimal ratio of 2.5 μl HilyMax:1 μg plasmid.

Animals ; Cell Line ; Chick Embryo ; Fibroblasts ; cytology ; Green Fluorescent Proteins ; genetics ; Plasmids ; genetics ; Transfection ; methods