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

OBJECTIVETo investigate endostatin gene therapy of rat corneal neovascularization induced by acid cauterization.

METHODSpBlast-hEndostatin and pBlast-Mcs were identified by digestion with Nhe Iand Sal I, by PCR reaction, by sequence, and then by alignment of PCR products with the gene Bank using NCBIBLAST software. They were then purified with QIAGEN Endofree plasmid maxi kit. Rat corneal neovascularization models were made with 75% AgNO(3) and 25% KNO(3) cauterization. The treatment method was subconjunctive injection of the pBlast-hEndostatin with the control of pBlast-Mcs.

RESULTSpBlast-hEndostatin was found to contain the human endostatin gene. The rat corneal neovascularization induced by acid cauterization was significantly suppressed after subconjunctive injection of the pBlast-hEndostatin with inhibition rates of 37%, 40.2%, and 42.8% respectively on the sixth, tenth, and fifteenth day. The inhibition rate for the density of corneal neovascularization was 40%. However, no inhibition effect on the length of the neovascularization and corneal inflammatory cells was observed. Corneal neovascularization areas were positively correlated with edema and corneal opacity.

CONCLUSIONSThe plasmid of pBlast-hEndostatin contained the human endostatin gene. The rat corneal neovascularization induced by acid cauterization can be partially inhibited by subconjunctive injection of the pBlast-hEndostatin mediated by liposomes. Endostatin produced by transfected fibroblast cells directly inhibits corneal neovascularization. This is not caused by inflammatory reaction inhibition.

Animals ; Corneal Neovascularization ; pathology ; therapy ; Endostatins ; genetics ; Genetic Therapy ; methods ; Humans ; Rats ; Rats, Wistar ; Transfection

AIMTo determine if the diagnostic ultrasound and self-made microbubbles could be used to increase gene transfection and expression in cardiac myocytes by means of the ultrasound-mediated microbubbles destruction.

METHODSThe perfluoropropane-exposed sonicated dextrose albumin(PESDA) microbubbles were made and mixed with indicated volume reporter gene encoding beta-galactosidase prior to gene transfection. Gene transfection into the cultured cardiac myocytes was performed by exposure to the various intense diagnostic ultrasound (1.3 MHz) in the presence of the gene-attached microbubbles. The calcium phosphate precipitation gene transfection was carried out alone or in combination with ultrasound-mediated destruction microbubbles. The cells were harvested 48 h after transfection and beta-galactosidase expression was detected by in situ staining and quantitive assay.

RESULTSCardiac myocytes exposed to ultrasound with PESDA induced significantly increase in gene expression (60-fold compared with naked plasmids transfection, P < 0.01). Moreover, it was found that the reporter gene expression not only related with ultrasound intension but also with the microbubbles concentration. In combination with calcium phosphate precipitation gene transfection, ultrasound-mediated destruction microbubbles resulted in more intense gene expression even 6 hours after calcium phosphate precipitation gene transfection.

CONCLUSIONThe ultrasonic destruction of gene-loaded microbubble is a highly effective gene transfer method, and it not only acts on the gene entry into cells, but also on the intracellular exogenous DNA expression.

Animals ; Gene Expression ; Genes, Reporter ; Myocytes, Cardiac ; cytology ; Plasmids ; Rats ; Rats, Wistar ; Transfection ; methods ; Ultrasonics

AIMTo study polyethylenimine (PEI)-mediated in vivo gene transfection into testis cells and preliminary functional research of spermatogenic cell-specific gene NYD-SP12 using this method.

METHODSPEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcellular localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining.

RESULTSWith the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells (especially in primary spermatocytes). Transfection into Sertoli cells was not observed. The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis.

CONCLUSIONPEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our study, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes.

Animals ; Green Fluorescent Proteins ; Homeodomain Proteins ; genetics ; physiology ; Humans ; Male ; Mice ; Polyethyleneimine ; Spermatogenesis ; physiology ; Transfection ; methods

OBJECTIVETo evaluate the feasibility of using iron oxide nanoparticles as gene vector and the effect of magnetic field on efficiency of transfection.

METHODSIron oxide nanoparticles were prepared by alkaline precipitation of divalent and trivalent iron chloride. The surface of iron oxide nanoparticles was modified by self-assembled poly-L-lysine to form particle complexes (IONP-PLL). Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using IONP-PLL as vector. The effect of magnetic field on efficiency of transfection was determined using Nd-Fe-B permanent magnet.

RESULTSForeign gene could be delivered to various cell lines by IONP-PLL and expressed with high efficiency, but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5 - 10 fold.

CONCLUSIONIONP-PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

Animals ; COS Cells ; Ferric Compounds ; administration & dosage ; Genetic Vectors ; Magnetics ; Polylysine ; administration & dosage ; Transfection ; methods

To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.
Cell Line, Tumor ; Green Fluorescent Proteins ; Humans ; Microbubbles ; Oligodeoxyribonucleotides, Antisense ; genetics ; Sulfur Hexafluoride ; Transfection ; methods ; Ultrasonics

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 observe the effect of plasmids in different size and gene transfection protocol on efficiency of introducing gene into human KC.

METHODSFour plasmids in different size, inclu-ding pSUPER-enhanced green fluorescent protein (EGFP), pEGFP-N2, pHSER-green fluorescent protein (GFP) and ploxP-EGFP, were transfected into immortal human KC line (HaCaT) and human embryo kid-ney cell line (293FT) separately following transfection protocols of liposome (LTP), cation polymerizer (CPTP), electroporation combined with nucleus transfection agent (ETP) and lentivirus. 293FT was used as control. GFP expression was observed under inverted fluorescence microscope. The transfection efficiency (TE) was calculated.

RESULTS(1) The four plasmids could be introduced into HaCaT (TE, 1.0%-3.3%) and 293FT (TE, 80.0%-84.7% ) following LTP. (2) The four plasmids could also be introduced into HaCaT (TE, 1.0%-3.7% ) and 293FT (TE, 81.3%-86.7% ) following CPTP. (3) Two shorter plasmids (pSUPER-EGFP and pEGFP-N2) could be introduced into HaCaT by ETP with higher TE than the othr two longer plasmids (pHSER-GFP and ploxP-EGFP), which were 22.3% and 19.0% vs. 4.0% and 3.3%, respectively. (4) pHSER-GFP packaged by lentivirus could be introduced into HaCaT with the TE reaching 97.0%, which surpassed the above three protocols.

CONCLUSIONSIt is difficult to introduce exogenous gene into human KC by LTP or CPTP; TE of lentivirus transfection protocol apparently surpasses

Cell Line ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Keratinocytes ; Liposomes ; metabolism ; Plasmids ; Transfection