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At least 109 records · Page 6Linked to original sources
PubMed2000

Adventitial versus intimal liposome-mediated ex vivo transfection of canine saphenous vein grafts with endothelial nitric oxide synthase gene.

PURPOSE: Experiments were designed (1) to evaluate liposome-mediated endothelial constitutive nitric oxide synthase (ecNOS) transfection in vein grafts and (2) to compare intimal and adventitial routes of transfection. METHODS: Male mongrel dogs (N = 36) underwent bilateral femoral artery bypass grafting with the lateral saphenous vein. In each animal one vein was transfected with plasmid (pVR1012) containing the ecNOS gene, and another vein was transfected with plasmid alone (control). Gene transfer was performed from either the intimal surface (Group I, n = 18) or the adventitial surface (Group II, n = 18). In each group there were three transfection subgroups (n = 6 each): (a ) 10 microg/mL naked plasmid DNA, (b ) 10 microg/mL plasmid DNA + liposome (LipofectAMINE PLUS), and (c ) 100 microg/mL plasmid DNA + LipofectAMINE PLUS. Grafts were harvested on the third postoperative day, and the transfection was assessed with molecular techniques and enzyme assay for activity of NOS by conversion of tritiated l-arginine to tritiated l-citrulline. Proliferating cells were quantified with a digital analysis of histologic sections after nuclear antigen Ki-67 (MIB1) immunohistochemistry. RESULTS: Transgene was identified with polymerase chain reaction in all ecNOS-transfected grafts, regardless of transfection modality. However, significant transcription of the ecNOS transgene was observed only in Group IIc (mean ecNOS messenger RNA, 8.7+/-1.7 vs. 3.1+/-0.7 x 10(-2) attomole/microL, in transfected compared with control grafts, respectively, P =.01). NOS activity increased approximately twofold in this group (11.58+/-2.1 and 6.3+/-1.0 pmol tritiated l-citrulline per milligram protein per hour in transfected and control grafts, respectively, P = .05). Numbers of proliferating cells did not differ among ecNOS-transfected and control grafts in any transfection group. CONCLUSION: These results suggest that ecNOS transfection of vein grafts is feasible through intimal and adventitial routes with naked DNA or a liposomal vector. However, efficient transcription of the transgene is evident at postoperative day 3 only after adventitial transfection of 100 microg/mL of the gene.

Animals↗
PubMed2005

Optimization of high-efficiency transfection of adult human mesenchymal stem cells in vitro.

With the advent of recent protocols to isolate multipotent human mesenchymal stem cells (MSCs), there is a need for efficient transfection methodologies for these cells. Most standard transfection methods yield poor transfection efficiencies for MSCs (<1%). Here we have optimized a high-efficiency transfection technique for low passage MSCs derived from adult human bone marrow. This technique is an extension of electroporation, termed amaxa Nucleofection, where plasmid DNA is transfected directly into the cell nucleus, independent of the growth state of the cell. With this technique, we demonstrate up to 90% transfection efficiency of the viable population of MSCs, using plasmid construct containing a standard cytomegalovirus (CMV) early promoter driving expression of green fluorescent protein (GFP). Although little variation in transfection efficiency was observed between patient samples, a 2-fold difference in transfection efficiency and a 10-fold difference in expression levels per cell were seen using two distinct CMV-GFP expression plasmids. By fluorescence-activated cell sorting, the GFP expressing cells were sorted and subcultured. At 2 wk posttransfection, approx 25% of the population of sorted cells were GFP positive, and by 3 wk, nearly 10% of the cells still retained GFP expression. Transfection of these cells with plasmid containing either the collagen type I (Col1a1) promoter or the cartilage oligomeric matrix protein (COMP) promoter, each driving expression of GFP, produced a somewhat lower transfection efficiency (approx 40%), due in part to the lower activity of transcription from these promoters compared to that of CMV. Transfection with the collagen type II (Col2a1) promoter linked to GFP exhibited low expression, due to the fact that collagen type II is not expressed in these cells. Upon culturing of the Col2a1-GFP transfected cells in a transforming growth factor-beta3-containing medium known to induce mesenchymal chondrogensis, a significant enhancement of GFP level was seen, indicating the ability of the transfected cells to differentiate into chondrocytes and express cartilage-specific genes, such as Col2a1. Taken together, these data provide evidence of the applicability of this technique for the efficient transfection of MSCs.

Adult↗
PubMed1990

Genetic instability and the acquisition of metastatic ability by rat mammary cancer cells following v-H-ras oncogene transfection.

When nonmetastatic dimethylbenzanthracene-induced rat mammary cancer cells (RMC1) were transfected with a control plasmid containing the neomycin resistance gene (i.e., Neo/Only), none of the five clonal Neo/Only transfectants isolated and characterized was metastatic, only one of five had a single structural chromosomal abnormality, and only one of five had a single numerical chromosomal change not present in the untransfected parental RMC1 cells. In contrast, when RMC1 cells were transfected with a plasmid containing both the neo resistance and mutated v-H-ras oncogene (i.e., Neo/Ras), four of nine clonal Neo/Ras transfectants isolated and characterized were highly metastatic, and all nine had multiple structural and/or additional numerical chromosomal abnormalities. The frequency of transfectants which had structural and/or additional numerical chromosomal changes in Neo/Ras transfectants was significantly higher than that in Neo/Only transfectants (P less than 0.05). In addition, seven of nine Neo/Ras transfectants had structural abnormalities in chromosome 1, whereas none of five Neo/Only transfectants had such abnormalities (P less than 0.05). All four Neo/Ras transfectants that were highly metastatic had structural aberrations involving a gain in chromosome 4. In contrast, none of the three Neo/Ras transfectants which were of low metastatic ability had a similar aberration involving chromosome 4. Correlation between a gain in chromosome 4 and a gain of high metastatic ability was significantly (P less than 0.05). These results demonstrate that, when RMC1 cells are transfected with v-H-ras, transfectants expressing the mutated v-H-ras p21 become genetically unstable and undergo chromosomal changes. These studies suggest that, if the appropriate chromosomal changes occur, these v-H-ras transfectants acquire high metastatic ability.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Transfer by pro gene transfection of tumor promoter-sensitive phenotype to promotion-insensitive JB6 cells.

Transfection of activated promotion sensitivity genes (pro genes) confers on insensitive (P-) cells susceptibility to induction of anchorage-independent growth by tumor-promoting phorbol esters. Promotion-sensitive (P+) JB6 cell variants, from which activated pro-1 and pro-2 were cloned, respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and various nonphorbol tumor promoters with anchorage-independent transformation that is irreversible 60-80% of the time. Anchorage-independent (Tx) clonal lines derived from these TPA-induced agar colonies were also tumorigenic in nude mice. This report has addressed the question of whether the phenotypes associated with parental P+ cells are transferred by transfection of activated pro-1 and pro-2. Clonal lines were established after transfection of JB6 P- cells with activated pro-1 or pro-2, induction of anchorage-independent colony formation by TPA, and growth of individual agar colonies to yield clonal transfectant lines. The lines so derived from transfected populations included Tx, P+, and P- lines, reflecting irreversible neoplastic transformation and greater and lesser degrees of preneoplastic progression, respectively. The anchorage-independent transfectants were found to be tumorigenic. Since untransfected P- cells subjected to the same single-cycle TPA treatment and cloning in agar yielded no anchorage-independent and few P+ transfectants, the appearance of P+ and Tx transfectants after pro-1 and pro-2 transfection is therefore likely to be due to the transfected pro genes. Indirect assay of pro gene uptake by quick-blot hybridization of transfectant cell DNA with the vectors into which pro genes had been cloned confirmed the association of transferred P+ and Tx phenotypes with the presence of the transfected DNA. Finally, assay of the sensitivity of P+ pro-1 and pro-2 transfectants to transformation by TPA at various concentrations showed that transfection with pro-1 or pro-2 conferred about equal responses that were somewhat lower than those observed with parental P+ controls. Taken together these data indicate that promotion-insensitive JB6 cells need only an activated pro gene and TPA exposure to become neoplastically transformed.

Animals↗
PubMed2004

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action.

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.

Animals↗
PubMed1998

A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages.

Primary cultured human macrophages are difficult to transfect. We have developed a DEAE-dextran DNA transfection method that mediates the reproducible transfection of primary cultured adherent human macrophages. Three factors essential for successful transfection were identified: DEAE-dextran concentration, the quantity of DNA per transfection and the incubation time of the macrophages with the transfection medium. Maximum levels of luciferase expression were attained within 24 h and maintained for at least 56 h after transfection. While serum in the transfection medium attenuated transfection, the treatment of the macrophages with chloroquine, DMSO, or glycerol did not enhance transfection within this system. A CMV enhancer/promoter mediated substantially greater luciferase expression in the macrophages than either HIV or RSV LTRs. DEAE-dextran facilitated superior transfection compared to either cationic liposome and calcium phosphate methods, and was more practical compared to electroporation for multiple transfections. This transfection protocol provides a simple, inexpensive, reproducible method for the evaluation of gene expression in primary cultured adherent human macrophages.

Adult↗
PubMed1996

Ultrasound-mediated transfection of mammalian cells.

Mammalian cells were successfully transfected with plasmid DNA in vitro using ultrasound transmitted through the walls of cell culture flasks or plates. Primary rat fibroblasts or chondrocytes were exposed to ultrasound in the presence of plasmids containing lacZ or neo genes. The transfection efficiency was evaluated by counting the number of beta-galactosidase (beta-Gal) positive cells or neomycin-resistant colonies. Transfection efficiency was optimized by varying ultrasound conditions, ambient temperatures (room temperature or 37 degrees C), plasmid concentrations, and initial cell populations. Additional experiments were performed performed to elucidate the mechanism of the ultrasound-mediated transfection. Maximal gene transfection was seen with two ultrasound conditions: 1-MHz carrier frequency 411 +/- 189 kPascal continuous wave with 20 or 30 sec of exposure time, and 1 MHz carrier frequency 319 +/- 157 kPascal continuous wave with 40 or 60 sec of exposure time. Gene expression was negligible when transfection procedures were performed at room temperature. The average stable transfection rate was 0.34% of surviving cells with a plasmid concentration of 40 micrograms/ml in primary fibroblasts. The transient transfection rate was 2.4% of surviving cells for primary chondrocytes. Data suggest that increasing plasmid concentration will increase efficiency. Identical treatment with 3.5 MHz produced no transfection, implying that cavitation produced by the ultrasound pressure wave appeared to play a critical role in mediating transfection. Ultrasound-mediated transfection was effective for suspended cells as well as for plated cells. This transfection method is simple, easy to keep sterile, and convenient. Ultrasound-mediated transfection appears to be a promising method for gene transfer into mammalian cells.

Animals↗
PubMed2003

Squamous metaplasia induced by transfection of human papillomavirus DNA into cultured adenocarcinoma cells.

BACKGROUND/AIM: It has been reported previously in cases of adenosquamous carcinoma of the lung in Okinawa, a subtropical island 2000 km south of mainland Japan, that the squamous cell carcinoma components were positive for human papillomavirus (HPV) by non-isotopic in situ hybridisation (NISH). The adenocarcinoma cells adjacent to the squamous cell carcinoma components were enlarged and also positive for HPV. This is thought to indicate that after adenocarcinoma cells are infected with HPV, they undergo morphological changes, and that "squamous metaplasia" follows. In this present study, the effects of HPV transfection into adenocarcinoma cells were examined. The relation between the region expressing the HPV gene and squamous metaplasia was also studied. METHODS: Plasmid pBR322 containing HPV type 16 (HPV-16) was transfected into cultured colonic adenocarcinoma (DLD-1) and lung adenocarcinoma (PC-14) cells using the calcium phosphate method. Neomycin was used as a selection marker. The presence of HPV E1, E2, E4, E5, E6, E7, L1, and L2 mRNAs and also transglutaminase 1, involucrin, cyclin dependent kinases (CDKs), cyclins, caspases, apoptosis inducing factor, DNase gamma, Fas, and Fas ligand mRNAs in HPV transfected cells was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The G0-G1 cell population was analysed by flow cytometry. Morphological examination under light and electron microscopes was also carried out. RESULTS: The virus transfected cells showed squamous metaplasia when they were injected into severe combined immunodeficient mice, expressing the high molecular weight keratin (Moll's number 1 keratin) and involucrin molecules immunohistochemically, and involucrin and transglutaminase I mRNAs by RT-PCR. The squamous metaplasia was most conspicuous in the HPV transfected DLD-1 cell when compared with HPV transfected PC-14 cells. Squamous metaplasia was most clearly demonstrated in one HPV transfected DLD-1 cell clone, which expressed not only E2 but also E6-E7 fusion gene mRNA. Viral L1 mRNA expression was absent in HPV transfected cell clones, and was not related to squamous metaplasia. The growth rate of HPV transfected cells was reduced. Transfection of the virus into the cultured adenocarcinoma cells increased the G0-G1 cell population greatly, as assessed by flow cytometer analysis. Furthermore, in the virus transfected cells, apoptosis was also observed by means of the terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling method. CONCLUSION: HPV transfection into adenocarcinoma cells induced clear squamous metaplasia. One of the HPV transfected cell clones that expressed E2 and E6-E7 fusion gene mRNA showed the squamous metaplasia particularly clearly, and apoptosis was also demonstrated.

Adenocarcinoma↗
PubMedDate not supplied

Liposome-mediated high-efficiency transfection of human endothelial cells.

Liposome-mediated transfection of endothelial cells provides a valuable experimental technique to study cellular gene expression and may also be adapted for gene therapy studies. However, the widely recognized disadvantage of liposome-mediated transfection is low efficiency. Therefore, studies were performed to optimize transfection techniques in human endothelial cells. The majority of the experiments were performed with primary cultures of human umbilical vein endothelial cells (HUVEC). In addition, selected experiments were performed using human brain microvascular endothelial cells and human dermal microvascular endothelial cells. To study transfection rates, HUVEC were transfected with the pGL3 vector, containing the luciferase reporter gene, complexed with several currently available liposomes, such as different Perfect Lipid (pFx) mixtures, DMRIE-C, or lipofectin. The optimal transfection rate was achieved in HUVEC transfected for 1.5 h with 5 microg/ml of DNA plasmid in the presence of 36 microg/ml of pFx-7. In addition, transfection with the VR-3301 vector encoding for human placental alkaline phosphatase revealed that, under the described conditions, transfection efficiency in HUVEC was approximately 32%. Transfections mediated by other liposomes were less efficient. The usefulness of the optimized transfection technique was confirmed in HUVEC transfected with NF-kappaB or AP-1-responsive constructs and stimulated with TNF or LPS. We conclude that among several currently available liposomes, pFx-7 appears to be the most suitable for transfections of cultured human endothelial cells.

Alkaline Phosphatase↗
PubMed2002

[Recombinant heat shock protein 70 adenovirus transfection protects intestinal epithelial cells (IEC-6 cells) against hypoxia-reoxygenation in vitro].

OBJECTIVE: To study the feasibility of protection of intestinal epithelial cells (IEC-6 Cells) against hypoxia-reoxygenation through recombinant adenovirus-mediated heat shock protein 70 (HSP70) gene transfection. METHODS: Recombinant adenovirus (AdCMVHSP70) was constructed with full length human HSP70 gene. Cultured intestinal epithelial cells (IEC-6 cells) were divided into four groups: three groups transfected with AdCMVHSP70 for 24 h, 48 h and 72 h respectively, and one group transfected with vacant recombinant adenovirus (control group). The over expression of transfected human HSP70 gene was detected by RT-PCR 48 h after transfection. After the cells in the four groups suffered 1 hour of hypoxia followed by 1 hour of reoxygenation, the effects of transfected human HSP70 gene overexpression during different transfection times (24 h, 48 h, and 72 h) were studied. The cell viability was analyzed by MTT method, and the apoptosis and dead cell ratio were evaluated with Annexin-V-FLOUS staining kit in vitro. RESULTS: The expression of human HSP70 gene was positive in the AdCMVHSP70 transfected IEC-6 cells and negative in the control group. After hypoxia-reoxygenation, the cell viability rates of HSP70 gene transfected groups were 87.24 +/- 0.60, 90.27 +/- 0.64, and 78.52 +/- 0.61 respectively, all significantly higher than that of control group (all P < 0.01), while the apoptosis rates of HSP70 gene transfected 24 h and 48 h groups were 3.93 +/- 0.28 and 3.95 +/- 0.54 respectively, significantly lower than that of control group (both P < 0.05), however, the apoptosis rate of HSP70 gene transfected 72h group was 11.49 +/- 1.45, not significantly different from that of control group (11.39 +/- 1.62, P > 0.05). The dead cell ratios of HSP70 gene transfected groups were 8.21 +/- 0.26, 5.83 +/- 0.32, and 5.83 +/- 0.32 respectively, significantly lower than that of control group (15.51 +/- 0.46, all P < 0.01). The peak of protection was at 48 h after the transfection. CONCLUSION: The over expression of human HSP70 mediated by recombinant adenovirus protects intestinal epithelial cells (IEC-6 cells) against hypoxia-reoxygenation in vitro. The probable mechanism may be concerned with the inhibition of cell apoptosis after hypoxia-reoxygenation.

Adenoviridae↗
PubMed2002

[Culture of human fibroblasts transfected by human telomerase reverse transcriptase eucaryotic expression plasmid pGRN145 and their biological characteristics in vitro].

OBJECTIVE: To analysis the biological characteristics of human fibroblasts transfected by human telomerase reverse transcriptase (hTERT) eucaryotic expression plasmid pGRN145. METHODS: Fibroblasts from children's foreskin were isolated and cultured in vitro, and the fibroblasts were transfected by pGRN145 with Lipofec-tAMINE PLUS Reagent. After strict screening of hygromycin B, the positive clones were subcultured. The telomerase activity was detected by RT-PCR and TRAP-PCR technique. The cell generation cycle and apoptosis rate were detected by flow cytometry to investigate the proliferative characteristics after transfection, and the chromosome karyotype of transformed cells was analyzed. The collagen secreted by transformed cells was detected by immunohistochemical staining. RESULTS: The morphological properties of fibroblasts did not change obviously after transfection. There were telomerase activity in transfected fibroblasts, while it could not be detected in pre-transfection fibroblasts. The cell generation cycle had no obvious changes between pre-transfection and post-transfection. However, the apoptosis rate of transfected fibroblasts were decreased compared with that of pre-transfection. The fibroblasts transfected by pGRN145 maintained the normal diploid karyotype, as well as the cells could normally secret type I and III collagen. CONCLUSION: The human fibroblasts transfected by pGRN145 has telomerase activity with prolonged life span of culture, which preliminarily proves the availability of establishing standard seeding cell lines of tissue engineering by hTERT plasmid transfection techniques.

Apoptosis↗
PubMed1996

Transfection alters ion transport in MDCK cells.

In the course of an investigation into the effect of Tamm-Horsfall protein (THP) on ion transport, we performed stable transfection of THP into MDCK cells using the SV40 or the cytomegalovirus (CMV) promoter. As controls, we transfected MDCK cells with an "empty" plasmid containing SV40 or CMV promoter but without THP cDNA. In another set of controls, we subjected cells to transfection procedures without DNA (mock transfection). K influx was not altered in cells subjected to mock transfection procedures without DNA, but both ouabain sensitive (OS) and ouabain resistant (OR) components of K influx were diminished in cells transfected with THP cDNA using either SV40 or CMV promoter. However, K influx was also reduced in cells transfected with a control plasmid containing either the SV40 promoter alone, or the CMV promoter alone, without the THP cDNA. Thus, the transport alterations were caused by transfection and not by THP. The reduction in ouabain-sensitive K influx was accompanied by a proportional reduction in the abundance of Na-K pump units as assessed by [3H] ouabain binding. [3H] bumetanide binding, a measure of the number of functioning NaK2Cl cotransporter sites, was reduced pari passu with the reduction in bumetanide-sensitive K influx. These results highlight the possibility that alterations in properties of transfected cells may not be solely due to the presence of transfected protein, but the result of some process associated with transfection itself. Without appropriate controls to evaluate this possibility, results of transfection studies are subject to potentially faulty and misleading interpretation.

Cell Line↗
PubMed2002

The role of the cell cycle on the efficiency of photochemical gene transfection.

The efficiency of gene transfection mediated by nonviral vectors is limited because of nonoptimal intracellular trafficking of transfecting DNA. Most nonviral vectors deliver transfecting DNA into a cell through endocytosis. However, poor escape from endocytic vesicles and inefficient transport of DNA into the nucleus often limits a success of gene transfection. Photochemical transfection is a new method, based on light-induced permeabilisation of endocytic vesicles, liberating transfecting DNA into the cytosol, concurrently increasing the chances for DNA to enter the nucleus. The aim of this study was to investigate the role of the cell cycle for the efficiency of photochemical transfection. It was demonstrated that in asynchronous human colon carcinoma HCT 116 cells photochemical treatment increased the transfection mediated by the nonviral vectors, the cationic polypeptide polylysine and the cationic lipid N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (beta AE-DMRIE/DOPE), by 30- and 2.5-fold, respectively. In aphidicolin-synchronised cells, photochemical transfection mediated by polylysine was dependent on the cell cycle: transfection level was 4-fold higher when illumination, inducing photochemical reactions, was performed during the G2/M phase as compared to the G1/early-S phase. The cell cycle influence on photochemical transfection mediated by beta AE-DMRIE/DOPE was very low: only 20% difference between G2/M and the G1/S phase was observed. We suggest that transgenes, photochemically liberated close/during mitosis, perhaps have the highest opportunity to enter the nucleus and be expressed. However, the dependence of photochemical transfection on the cell cycle might be partially disguised by various factors induced by photochemical treatment.

Aphidicolin↗
PubMed2000

Efficient in vitro transfection of human keratinocytes with an adenovirus-enhanced receptor-mediated system.

Efficient gene delivery to the skin is important for gene therapy of skin diseases and in-depth biologic studies of epidermis. In this report, we investigated three nonviral transfection systems for gene transfer in cultured human keratinocytes and organotypic cultures. SuperFect is a highly branched polycationic transfection reagent, PrimeFector a polycationic liposome compound, and the AVET (adenovirus-enhanced transferrin-mediated) system consists of a ternary complex of biotinylated chemically inactivated adenovirus noncovalently complexed with plasmid DNA and polylysine-transferrin. After AVET transfection of cultured keratinocytes with pCIbetagal, a CMV/beta-galactosidase reporter plasmid, 28.8% +/- 1.4% of the cells were stained blue. SuperFect was about 2-fold less efficient, whereas Primefector did not transfect keratinocytes. Similar results were obtained when transfection efficiencies were measured by enzyme assays. Addition of holotransferrin to the culture medium or replacement of polylysine-transferrin by polylysine in the ternary complex did not affect the transfection efficiency. Using AVET complexes without adenovirus, however, strongly diminished gene delivery. This indicates that the AVET complex is taken up by an adenovirus receptor. Separation of AVET/pCIbetagal transfected keratinocytes by adhesion to collagen IV into two fractions (rapidly and slowly adhering cells) showed that the latter were transfected at a 3-fold higher level. Therefore, it seems that putative stem cells adhering rapidly to collagen IV are not efficiently transfected by AVET. AVET-transfected keratinocytes derived from keratinocyte trans- glutaminase negative lamellar ichthyosis patients with a CMV-TGK expression plasmid showed that it is possible to reach a level of total enzyme activity similar to that found in cultured keratinocytes from normal individuals. In organotypic cultures from outer root sheath cells AVET transfection was not successful, which might be due to the presence of the cornified layer or inaccessibility of the adenovirus receptor. In summary, the AVET system provides a powerful tool for transient in vitro transfection of keratinocytes.

3T3 Cells↗
PubMed2002

Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes.

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.

Animals↗
PubMed1998

Prolonged survival of rat liver allografts transfected with Fas ligand-expressing plasmid.

BACKGROUND: Transplantation of Fas ligand (FasL) gene-transfected tissues can have opposite effects. For example, cotransplantation of pancreas islets with myoblasts transfected with FasL-expressing plasmid vector (pFasL) prevented graft rejection, whereas the expression of FasL directly within islets using adenovirus vector led to graft destruction. It was also reported that FasL expression on pancreas islets led to neutrophilic infiltration and rapid destruction of the islets. From these results, overexpression of FasL in transfected tissues may lead directly to self destruction through an autocrine Fas-FasL pathway or graft destruction through neutrophil recruitment. To date there have been no reports of successful transplantation of FasL gene-transfected solid organs. METHODS: Rat pFasL was transfected at a dose of 90, 180, 270, or 360 microg into rat liver with an inactivated hemagglutinating virus of Japan conjugated to liposome vesicles (HVJ-liposome), and the gene-transfected livers were transplanted to allogeneic rats. RESULTS: In 18 rats transfected with 180 microg of pFasL, 14 (78%) did not develop fulminant hepatitis. FasL-mRNA was detected in these livers at 3, 5, 7, and 14 days after transfection. The expression of FasL protein was also observed in the transfected liver, and the transfection rate by this method was 11.1+/-1.9%. The livers were then transplanted to allogeneic recipients, resulting in significant (P<0.01) prolonged recipient survival times. Histological observation showed that the pFasL-transfected liver allografts caused apoptotic cell death in infiltrating activated T cells. In contrast, transfection of pFasL higher than 180 microg resulted in lethal hepatitis in all rats, and its low dose (90 microg) did not induce the hepatitis or prolong recipient survival. CONCLUSION: Our results indicate that rat liver allografts can be protected to host immune responses by an adequate level (approximately 10%) of FasL expression in the livers using HVJ-liposome incorporating pFasL.

Animals↗
PubMed2002

Co-transfection of p16(INK4a) and p53 genes into the K562 cell line inhibits cell proliferation.

BACKGROUND AND OBJECTIVES: The tumor suppressor genes p53 and p16(INK4a), both of which act in tumor surveillance, are homozygously deleted in the human leukemia cell line K562. This study was performed to assess whether co-transfection of the p16(INK4a) and p53 genes could inhibit K562 cell proliferation. DESIGN AND METHODS: p16(INK4a) and p53 genes were co-transfected into K562 cells with liposome, and the expression of the transfected genes was detected by Western-immunoblotting and immunocytochemistry. The effect of the p16(INK4a) and p53 transfected cell culture was quantified by trypan blue staining, and the number of recovered viable cells was assessed every day after transfection. Cells were analyzed for expression of annexin V in order to detect apoptosis. Differentiation of transfected K562 cells was measured by the benzidine oxidation test, and the cell cycle was analyzed by flow cytometry. RESULTS: After co-transfection, there were 23% and 28% p53 and p16(INK4a) positive cells respectively. Co-transfection with p16(INK4a) and p53 genes significantly inhibited cell proliferation when compared to transfection with either p16(INK4a) or p53 gene. The percentage of cells expressing the apoptosis-related cell surface antigen annexin V was significantly higher in p53 and p16(INK4a) transfected cells than in p53 or p16(INK4a) transfected cells (6.24+/-0.37% vs 4.88+/- 0.17%, p<0.05 and vs 2.78+/-0.26%, p<0.05, respectively). p16(INK4a) and p53 co-transfection significantly increased the number of cells in G1 phase and decreased that in S phase. INTERPRETATION AND CONCLUSIONS: Expression of wild-type p16(INK4a) and p53 genes in K562 cells results in reduced proliferation and apoptosis. Introduction of exogenous p16(INK4a) and p53 genes into K562 cells might contribute to the clinical treatment of leukemia.

Annexin A5↗
PubMed2005

[The effect of transfecting Stat3beta cDNA on human breast cancer].

OBJECTIVE: To study the effect of transfecting Stat3beta cDNA on human breast cancer. METHODS: Human breast cancer cells of the line SK-BR-3 were cultured and divided into 3 groups: Stat3beta transfection group (to be transfected with plasmid pIRES-Stat3beta containing Stat3beta by transient transfection technique), lipofectin reagent transfection group pIRES-EGFP transfection group, and control group. The positively transfected cells were isolated by fluorescence-activated cell sorter. Flow cytometry was used to analyze the cell cycles and cell apotosis. Western blotting was used to detect the expression of STAT3 protein. MTT method was used to examine the proliferation of the cells. RESULTS: Forty-eight hours after exposure to the plasmid pIRES-Stat3beta the transfection rate of the SK-BR-3 cells was 13.79%. SK-BR-3 cells expressed STA3 protein during proliferation. In comparison with the SK-BR-3 cells of other 3 group, the proliferation of the cells transfected with pIRES-Stat3beta was significantly decreased. Forty-eight hours after transfection, 81.09% of the cells transfected with the plasmid pIRES-Stat3beta accumulated at the G(0)/G(1) stage, a rate significantly higher than those of the other groups, and displayed a significantly higher rate of apoptosis. CONCLUSION: Transfection of plasmid pIRES-Stat3beta containing Stat3beta blocks the Stat3 pathway, thus inhibiting the proliferation and augment the apoptosis of human breast cancer cells and providing a novel gene therapy target.

Acute-Phase Proteins↗