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At least 91 records · Page 5Linked to original sources
PubMed2003

Enhancement of polyethylene glycol (PEG)-modified cationic liposome-mediated gene deliveries: effects on serum stability and transfection efficiency.

In this study, we modified cationic liposomes either by polyethylene glycol (PEG)-grafting or PEG-adding methods, and compared the physical properties of transfection complexes and transfection efficiency in-vitro and prolonged circulation in-vivo. The PEG-grafted transfection complexes were prepared by mixing plasmid DNA with PEG-grafted cationic liposomes, which were composed of DSPE-PEG 2000 and cationic lipids. The PEG-added transfection complexes were prepared by adding DSPE-PEG 2000 to the mixture of cationic liposomes and plasmid DNA. The particle sizes of the PEG-modified transfection complexes (approximately 200 nm) changed a little over 4 weeks compared with the conventional transfection complexes. In the presence of serum, the transfection efficiency of the conventional transfection complexes was lowered whereas the transfection efficiency of the PEG-modified transfection complexes was maintained. Moreover, the transfection efficiency of the conventional transfection complexes was significantly reduced when they were stored. However, the transfection efficiency was stable for the PEG-modified transfection complexes, even after two weeks of storage. Of the in-vitro transfection efficiencies, there was no difference between PEG-grafted and PEG-added transfection complexes. When the conventional, PEG-grafted, and PEG-added transfection complexes were administered into mice by the tail vein, the PEG-added transfection complexes showed a prolonged circulation of plasmid DNA compared with other transfection complexes. These results suggest that the PEG-added transfection complexes could be a useful non-viral vector because of their simplicity in preparation, enhanced stability and prolonged circulation compared with the conventional transfection complexes.

Cations↗
PubMedDate not supplied

Comparative transfection studies of human ovarian carcinoma cells in vitro, ex vivo and in vivo with poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes.

BACKGROUND: Poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) can be used successfully for in vitro transfection of different cell lines, including the OVCAR-3 human ovarian carcinoma cell line. The aim of this study was to investigate whether it is possible to transfect OVCAR-3 cells in vivo with polyplexes containing p(DMAEMA). METHODS: In order to understand the generally observed gap between in vitro and in vivo transfection, we gradually went from in vitro to in vivo transfection of OVCAR-3 cells, while keeping the exposure conditions the same, as far as possible. To find the reason for the negligible degree of in vivo transfection, in vitro cultured OVCAR-3 cells were transfected in the presence of peritoneal ascites fluid. Next, the influence of hyaluronic acid, one of the ascites components, on the transfection efficiency was studied. RESULTS: P(DMAEMA)-containing polyplexes can transfect OVCAR-3 cells in vitro with an overall transfection efficiency of 10%. Cells grown in vivo can be transfected ex vivo with p(DMAEMA)/plasmid complexes with an overall transfection efficiency of approximately 1-2%. When transfection complexes are injected i.p. into nude mice bearing OVCAR-3 cells in the peritoneal cavity, the degree of in vivo transfection efficiency achieved is negligible. In vitro cultured OVCAR-3 cells were also transfected with polyplexes in the presence of peritoneal ascites fluid. The results indicate that one or more components of ascites had a negative effect on the transfection efficiency of p(DMAEMA)-containing polyplexes. To elucidate which component(s) of ascites may have interfered, the influence of hyaluronic acid, one of the ascites components, on the transfection efficiency was studied. The outcome suggests that hyaluronic acid may have induced a negative effect on the transfection capability of p(DMAEMA)-containing polyplexes. CONCLUSION: P(DMAEMA) is an efficient transfectant in vitro and ex vivo. However, transfected cells were not detected in vivo which may be caused by a negative influence of components of the ascites fluid.

Animals↗
PubMed2002

[An in vitro model of hepatitis B virus gene replication and expression in primary rat hepatocytes transfected with circular viral DNA].

OBJECTIVE: To establish an in vitro model of hepatitis B virus (HBV) replication and expression in primary rat hepatocytes (PRH) transfected with circular viral DNA for further study on the interaction of HBV with hepatocytes. METHODS: Circular viral DNA containing complete HBV genome were transfected into PRH by electroporation (transfected group, about 4mug of circular viral DNA/1 10(7)cells). From day 1 to day 10 after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH were measured with IMX system. HBcAg was assayed with western blotting, immunol dot blotting and immunocytochemistry. Meanwhile, HBV S-mRNA and X-mRNA were tested with RT-PCR, and replicative intermediates of HBV DNA were analyzed by southern blotting and dot blotting. Moreover, Transmission electron microscopy was used if viral particles were produced in transfected rat hepatocytes. PRH electroporated only was used as control group. RESULTS: (1) Viral antigen production in transfected rat hepatocytes: HBsAg in cell lysates was positive. P/N values ranged from 4.83 to 85.69, and could be maintained for 10 days after transfection. The average P/N values was 18.239 27.459. Whereas, HBsAg was negative in the supernatants of transfected group (P/N values, negative<2.1). HBeAg in the supernatants and lysates of transfected hepatocytes all was negative (P/N values<2.1) during 10 days following transfection. HBcAg was only found positive in transfected hepatocytes by immunol dot blotting. (2) Detection of viral transcripts: transcription of HBV DNA was investigated by preparing total RNA from rat hepatocytes 2 days after transfection and looking for S-mRNA and X-mRNA by RT-PCR. Results showed S-mRNA positive, X-mRNA negative. (3) HBV DNA replication analysis: intracellular total DNA was extracted 2 days after transfection and analysed by southern blotting. All replicative DNA intermediates, including relaxed circular (rcDNA), covalently closed circular (cccDNA), and single-stranded (ssDNA) linear HBV DNA forms, were indicated. Dot blotting showed intracellular HBV DNA positive in transfected group during 10 days after transfection. However, viral particles were not found in transfected hepatocytes during 3 days after transfection. CONCLUSIONS: Circular HBV DNA transfected into primary adult rat hepatocytes could obtain continuous replication and stable expression of HBV surface antigen. This in vitro model has high reproducibility and stability, and is useful for directly studying the interaction of HBV with hepatocytes.

Animals↗
PubMedDate not supplied

Sensitivity of anticancer drugs in NIH3T3' cells transfected with oncogenes accompanied by pSV2neo vector.

NIH3T3 cells and NIH3T3 cell lines, which were transfected with several oncogenes accompanied by pSV2neo vector, were observed for their survival rate when treated with commonly used anticancer drugs. The survival rates in the cell line transfected with the pSV2neo vector only did not differ significantly from that of parental NIH3T3 cells against bleomycin, nimustine and adriamycin, but it was significantly more resistant to cisplatin and more sensitive to mitomycin C. Therefore, the survival rate in each transfectant was compared with that in the pSV2neo transfectant. The Val-12, v-Ha-ras, v-int-2, v-erbB or v-abl transfectants accompanied by pSV2neo vector were significantly more sensitive to cisplatin than transfection with pSV2neo vector only. The Val-12, v-Ha-ras, v-int-2 and v-abl transfectants were significantly more sensitive, and the v-erbB transfectant more resistant to bleomycin than the pSV2neo transfectant. The v-int-2, v-erbB, frg, v-raf and v-myc transfectants were more sensitive, and the v-Ha-ras transfectant more resistant to nimustine than the pSV2neo transfectant. The N-ras, v-Ha-ras, v-sis, v-int, v-abl and v-myc transfectants were significantly more sensitive to adriamycin than the pSV2neo transfectant. The v-sis and v-int-2 transfectants were more sensitive, and the c-Ki-ras, Val-12, v-erbB and the v-src transfectants more resistant to mitomycin C than the pSV2neo transfectant. Thus there was no relationship between the drug sensitivity and the location of oncogenes, but the transfection was associated with either increased or decreased sensitivity to a number of commonly used anticancer drugs. Therefore, it may be important to take into consideration or overexpression of oncogenes in cancer chemotherapy.

3T3 Cells↗
PubMed1992

Growth regulation of estrogen receptor-negative breast cancer cells transfected with complementary DNAs for estrogen receptor.

BACKGROUND: The growth of estrogen receptor (ER)-positive breast cancer cells is hormonally regulated, but the majority of breast cancers are ER negative and unresponsive to hormonal therapy. PURPOSE AND METHODS: To test whether hormonal control over replication can be re-established in ER-negative cells, we transfected ER-negative MDA-MB-231 (clone 10A) cells with sense and antisense constitutive ER expression vectors containing the gene for either wild-type or mutant ER linked to the gene for neomycin resistance aminoglycoside phosphotransferase (neo). A Northern blot analysis was done on total RNA from eight of the 10 transfectant clones produced to detect messenger RNA coding for ER and neo, and a Western blot analysis was done on protein extracted from the cells of one mutant and two wild-type ER sense transfectant clones to determine the molecular weight of the ER in transfectants. Levels of ER in transfectants were measured both by enzyme immunoassay and by ligand-binding methods. To ascertain whether the ER in wild-type and mutant sense transfectants was functional, we tested the effects of 17 beta-estradiol (E2) and/or an antiestrogen, ICI 164,384, on 1) ER-activated gene regulation (by transient transfection of these cells a second time with a reporter plasmid containing an estrogen response element linked to the chloramphenicol acetyl transferase [CAT] gene), 2) induction of progesterone receptor, 3) DNA replication, and 4) cell cycle kinetics. RESULTS: Messenger RNA coding for ER and for neo was detectable in both sense and antisense transfectant clones. Sense transfectants (both mutant and wild-type) expressed ER protein with a molecular weight similar to that found in ER-positive control cells. By the ligand-binding method high levels of ER were detected in both wild-type and mutant transfectants, although by the enzyme immunoassay method lower levels were detected in mutant transfectants. ER from both wild-type and mutant sense transfectants appeared functional, since E2 stimulated the expression of reporter-linked CAT and of progesterone receptor in these transfectants. E2 inhibited DNA replication in wild-type sense transfectants at a concentration of 10(-10) M and mutant sense transfectants at a concentration of 10(-8) M, and ICI 164,384 blocked this effect. CONCLUSION: ER-negative breast cancer cells stably transfected with either a mutant or wild-type ER gene regain hormonal responsiveness; however, E2 inhibits rather than stimulates cell growth. IMPLICATION: Reactivation of quiescent ER may provide a novel therapeutic approach for controlling ER-negative breast cancers.

Base Sequence↗
PubMed2001

Nonviral transfection of intact pancreatic islets.

Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-beta-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 microl) and DNA (3 and 6 microg). Transfection efficiency was quantified by microscopic evaluation of beta-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express beta-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 microl DNA and 18 microl DOTAP/ml (1:6 ratio), 6 microg DNA and 12 microl DOSPER/ml (1:2 ratio), or 6 microg DNA and 12 microl Lipofect-AMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8+/-0.6 (mean +/- SEM) for DOTAP-transfected islets compared with 8.4+/-0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1+/-0.5 and 3.4+/-0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.

Animals↗
PubMed1993

Direct myocardial transfection in two animal models. Evaluation of parameters affecting gene expression and percutaneous gene delivery.

BACKGROUND: Gene therapy represents a novel approach to the treatment of a variety of disease states. Direct injection of pure untreated DNA into skeletal and cardiac muscle is sufficient to perform gene transfer in vivo. Little information is available, however, regarding the extent to which individual parameters of direct gene delivery affect the efficiency of myocardial transfection. Moreover, the fact that all previously reported studies of successful myocardial transfection were performed using open chest surgery to visualize the heart for direct injection of DNA constitutes a potential limitation to clinical applications of myocardial gene transfer. The objectives of the present study were: (a) to determine the extent to which gene expression is altered by varying the amount of DNA and the volume of the delivery vehicle; (b) to study whether protracted expression of DNA persists after direct myocardial transfection; and (c) to test the feasibility of percutaneous, transthoracic myocardial injection of DNA. EXPERIMENTAL DESIGN: New Zealand white rabbits (N = 30) were transfected with the firefly (Photinus pyralis) luciferase reporter gene. Twenty-three rabbits were segregated into 5 groups according to the amount of DNA and volume of delivery vehicle injected. These rabbits were sacrificed at 5 days posttransfection to measure the level of luciferase gene expression. Three more rabbits were designated exclusively for in situ hybridization. Two rabbits were sacrificed at 5 and 6 months, respectively to evaluate long-term expression. Two additional rabbits were used as controls. Yucatan microswine (N = 4) were used to evaluate the feasibility of percutaneous, transthoracic myocardial transfection with beta-galactosidase or luciferase reporter genes. Microswine were sacrificed immediately post-sham transfection (N = 2), or 5 days postmyocardial transfection (N = 2). RESULTS: Among 11 rabbits, transfection with 10 micrograms of DNA (N = 4), 25 micrograms of DNA (N = 3), and 50 micrograms of DNA (N = 4) in 100 microL of injectate yielded a step-wise but statistically insignificant increase in luciferase activity. Among 16 rabbits, transfection with 50 micrograms of DNA in injectate volumes of 50 microliters (N = 4), 100 microliters (N = 4), 150 microliters (N = 4), and 300 microliters (N = 4) yielded a statistically significant (p < 0.05) increase in luciferase activity in those hearts transfected with 300 microliters compared with those hearts transfected with either 50 or 100 microliters. Luciferase activity at 5 and 6 months postmyocardial transfection was 7.14 and 68.8 Turner light units, respectively. In situ hybridization confirmed that myocytes represented the site of luciferase expression. Percutaneous myocardial transfection was successfully accomplished with both reporter genes. CONCLUSIONS: The results of the present study demonstrate that increasing the amount of DNA employed for direct myocardial transfection fails to produce a statistically significant increase in the level of gene expression. In contrast, increasing the volume of injectate used to directly transfect a constant amount of DNA (50 micrograms) produced significantly augmented expression of the reporter luciferase gene. Luciferase expression was detected 6 months posttransfection. Successful transfection after fluoroscopic-guided direct percutaneous delivery suggests that it may be feasible to deliver genetic material into the myocardium of patients in a similar fashion.

Animals↗
PubMed1999

Transtracheal gene transfection of donor lungs prior to organ procurement increases transgene levels at reperfusion and following transplantation.

BACKGROUND: Gene therapy's potential to modify donor organs to better withstand the process of transplantation has yet to be realized. To determine whether gene transfection is feasible to treat the early post-transplant injury of ischemia-reperfusion, we compared transfection of lungs in the donor prior to organ procurement with transfection of harvested ex vivo lungs in a rat single lung transplant model. METHODS: Lewis rats (donor transfection [DT]; n = 4) underwent transtracheal adenoviral-mediated transfection with 10(9) plaque forming unit of the beta-galactosidase reporter gene. Donor lungs were harvested following 6 hours of in vivo post-transfection ventilation, and then preserved for 6 hours at 4 degrees C prior to left single-lung transplantation. Ex vivo transfection was performed following organ retrieval; lungs were then preserved at 4 degrees C for 6 hours (EVT6h; n = 6) and 12 hours (EVT12h; n = 6) prior to transplantation. Lung transgene expression was measured by chemiluminescence at reperfusion, and at 2 hours following lung transplantation. RESULTS: Donor transfection lungs showed significantly higher levels of transgene expression as compared with EVT lungs at the time of reperfusion (DT = 3,408+/-1,301 relative light units/mg protein; EVT6h = 218+/-7; EVT12h = 213+/-26; p < 0.02) and at 2 hours after lung transplantation (DT = 2900+/-870; EVT6h = 62+/-27; EVT12h = 123+/-21; p < 0.005). Transgene expression measured in the heart, liver, kidney, and serum from DT rats demonstrated virtually no evidence of collateral transfection at 12 hours post-transfection (all <5.0). CONCLUSIONS: Gene transfection of donor lungs produces significantly higher levels of transgene expression in lungs at the critical time of reperfusion and in the early period following lung transplantation as compared to ex vivo transfection of cold preserved lungs. Transtracheal donor-lung transfection does not appear to result in collateral transfection of other transplantable organs. Local adenoviral-mediated transfection of the lungs is possible in the multiorgan donor prior to organ procurement and may provide the optimal strategy for gene therapeutic manipulations to address post-transplant ischemia-reperfusion injury.

Animals↗
PubMed2002

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle.

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.

Animals↗
PubMed2005

Unsatisfactory gene transfer into bone-resorbing osteoclasts with liposomal transfection systems.

BACKGROUND: Bone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells. Many of the details of osteoclast formation, activation and motility remain unsolved. Therefore, there is an interest among bone biologists to transfect the terminally differentiated osteoclasts and follow their responses to the transgenes in vitro. Severe difficulties in transfecting the large, adherent osteoclasts have been encountered, however, making the use of modern cell biology tools in osteoclast research challenging. Transfection of mature osteoclasts by non-viral gene transfer systems has not been reported. RESULTS: We have systematically screened the usefulness of several commercial DNA transfection systems in human osteoclasts and their mononuclear precursor cell cultures, and compared transfection efficacy to adenoviral DNA transfection. None of the liposome-based or endosome disruption-inducing systems could induce EGFP-actin expression in terminally differentiated osteoclasts. Instead, a massive cell death by apoptosis was found with all concentrations and liposome/DNA-ratios tested. Best transfection efficiencies were obtained by adenoviral gene delivery. Marginal DNA transfection was obtained by just adding the DNA to the cell culture medium. When bone marrow-derived CD34-positive precursor cells were transfected, some GFP-expression was found at the latest 24 h after transfection. Large numbers of apoptotic cells were found and those cells that remained alive, failed to form osteoclasts when cultured in the presence of RANKL and M-CSF, key regulators of osteoclast formation. In comparison, adenoviral gene delivery resulted in the transfection of CD34-positive cells that remained GFP-positive for up to 5 days and allowed osteoclast formation. CONCLUSION: Osteoclasts and their precursors are sensitive to liposomal transfection systems, which induce osteoclast apoptosis. Gene transfer to mononuclear osteoclast precursors or differentiated osteoclasts was not possible with any of the commercial transfection systems tested. Osteoclasts are non-dividing, adherent cells that are difficult to grow as confluent cultures, which may explain problems with transfection reagents. Large numbers of alphavbeta3 integrin on the osteoclast surface allows adenovirus endocytosis and infection proceeds in dividing and non-dividing cells efficiently. Viral gene delivery is therefore currently the method of choice for osteoclast transfection.

Antigens, CD34↗
PubMed2004

[Expression of estrogen receptor in different breast cancer cell lines transfected with estrogen receptor plasmid HEGO5 mediated by FuGENE6].

BACKGROUND & OBJECTIVE: Gene transfection is a major approach in studies of estrogen receptor (ER)expression in breast cancer cells,and ER's functional mechanism. This study was designed to detect the transfection efficiency of different human breast cancer cell lines mediated by FuGENE6 reagent, and ER expression in these cells after transfected with ER plasmid HEGO5. METHODS: Breast cancer cell lines were transfected with plasmid pEGFP-N1,and the transfection efficiency was measured by flow cytometry (FCM). The cell lines with high transfection efficiency were transfected with HEGO5,and then the expression of ER was determined by FCM and Western blot. RESULTS: There were obvious differences in transfection efficiencies of breast cancer cell lines MM-231, MM330, MM134 VI, MM175VII, MM157, MM361, MM436, MM453, UaCC812, UaCC893, BT-549, BT-20, HBL-100, Hs578t, MCF-7, T-47d, and ZR-75-1. Repetitious transfection test in HBL-100 cells showed that co-efficient of variation was 5.1. HEGO5 was transfected into the cells with high plasmid transfection efficiency (MCF-7, HBL-100, Hs578t, MM436, MM453, and BT-20), positive rates of ER in these cells ranged from 12.9% to 54.8%, corresponding to the pEGFP-N1 transfection efficiency, and the expression of ER detected by FCM were in accordance with those tested by Western blot. To some extent, the expression of ER in transfected cells was cell cycle specific. CONCLUSIONS: Transfection of breast cancer cell lines mediated by FuGENE6 reagent has a good repetition,and the results were stable and reliable. In the cells transfected with HEGO5,the expression of ER can be qualitatively detected by Western blot, and quantitatively detected by FCM.

Breast Neoplasms↗
PubMed2006

Transfection of primary cultures of rat hepatocytes.

Five different transfection reagents-calcium phosphate, TransFast Transfection Reagent, Superfect Transfection Reagent, Effectene Transfection Reagent, and Tfx-20--were compared for their ability to effectively transfect primary cultures of male rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and then cultured on Matrigel-coated plates for 24 h before transfection. The cells were transfected with either pGL3-Control or pGL3-Basic plasmids. The efficiency of transfection of each reagent was monitored using the dual luciferase reporter gene assay system. Superfect Transfection Reagent, Effectene Transfection Reagent and Tfx-20 were the most effective for the transfection of primary hepatocytes and gave comparable transfection efficiencies. Calcium phosphate was found to be the least effective transfection reagent and gave the most variable transfection results. Tfx-20 gave the least variable transfection results when different hepatocyte preparations were compared.

Animals↗
PubMed2006

Cell-specific enhancement of liposomal transfection by steroids in steroid receptor expressing cells.

The DNA transfer efficiency in liposomal versus viral transfection is very low, mainly due to an insufficient nuclear transport of the delivered DNA after its endocytotic uptake to the cell. Ligand activation of intracellular steroid receptors and their subsequent mobilization to the nucleus could result in a co-transport of DNA to the nucleus. The augmentation of nuclear transport of DNA after steroid addition might cause enhanced transfection efficiency. We used cell lines from gynecologic malignoma expressing steroid receptors, such as T47D and Mcf-7 breast cancer cell lines, as well as receptor-negative cell lines, such as Hec1A from endometrium carcinoma or the breast cancer cell line MDA-MB-231. The cells were transfected by the liposomal transfection agent Dotap with the gene for firefly-luciferase as a reporter gene and transfection efficiencies were determined in the luciferase assay. We compared the effect of the addition of cholesterol and steroids in different cell lines on the transfection efficiency. The addition of cholesterol to transfection agents led to an enhancement of the luciferase activity in all cell lines. Steroids enhanced the transfection efficiency only in receptor-positive cell lines. The transfection efficiencies of HEC-1A or MDA-MB-231 cells were not enhanced by steroids. A progesterone preincubation of receptor-positive T47D cells resulted in a decrease of progesterone receptors and afterwards the progesterone enhanced transfection was dramatically diminished. We presume that the transfection enhancement by steroids is dependent on increased nuclear import of the delivered DNA only in the presence of steroid receptors. Steroid enhancement of transfection is different from the benefit of cholesterol for transfection that acts on general cellular properties or the transfection complex as such. Liposomal transfection in combination with steroids might be useful for a cell-specific enhancement of gene transfer for example in gynecological malignoma where subgroups are expressing high levels of steroid receptors.

Blotting, Western↗
PubMed1991

H-ras expression, genetic instability, and acquisition of metastatic ability by rat prostatic cancer cells following v-H-ras oncogene transfection.

To study the relationship between metastatic ability, mutated H-ras expression, and genetic instability, a cloned, nonmetastatic rat prostatic cancer cell line (AT2.1) was transfected with the v-H-ras oncogene. The parental AT2.1 clone, 4 control transfectants (Neo/Only), and 9 v-H-ras transfectants (Neo/Ras) were characterized with regard to their H-ras content by using Southern, Northern, and Western blot analysis and their biological behavior in vivo. Following s.c. inoculation in syngeneic rats, all transfectants produced tumors. All 4 (Neo/Only) transfectants like the parental untransfected cell were non-metastatic. Six of 9 Neo/Ras transfectants were metastatic to the lungs and lymph nodes, while the other 3 Neo/Ras transfectants were not metastatic. There was no simple dose-response relationship between the level of v-H-ras integration, mRNA or p21 protein expression, and the development of metastatic ability by the Neo/Ras transfectants. Cytogenetic analysis demonstrated that the frequency of additional structural and/or additional numerical chromosomal changes among the Neo/Ras transfectants was significantly higher than that in the Neo/Only transfectants (P less than 0.05). Loss of chromosome 10 was observed in all of the Neo/Ras transfectants, whereas that was observed in only one of the 4 Neo/Only transfectants (P less than 0.05). There were no specific chromosomal changes, however, which were statistically correlated with the development of metastases in the Neo/Ras transfectants. These results demonstrate that development of the metastatic ability in AT2.1 cells is not a single-step reaction regulated by the level of H-ras expression alone, but rather a complex process requiring additional events. One of the additional events appears to be an increase in genetic instability and cytogenetic changes following v-H-ras transfection.

Animals↗
PubMed2004

Molecular mechanism of c-jun antisense gene transfection in alleviating injury of cardiomyocytes treated with burn serum and hypoxia.

To explore the molecular mechanism of c-jun antisense gene transfection in alleviating injury of cardiomyocytes treated with burn serum and hypoxia, burn serum was collected from Wistar rats inflicted with 30% third-degree burn of the total body surface area. The cardiomyocytes of neonatal Wistar rats were cultured and then treated with burn serum and hypoxia (a gas mixture containing 1% O2). The constructed c-jun antisense gene recombinant was transfected into the cardiomyocytes of neonatal Wistar rats. TdT-mediated d-utp nick end labeling (TUNEL) was adopted to examine cardiomyocyte apoptosis. Morphological changes of cardiomyocytes were observed under an optic-microscope and an electron-microscope. Expression of troponin T and beta-tubulin protein, c-jun protein, protein kinase Ca (PKCa), and c-jun N-terminal kinase (JNK) were assayed with Western blot in the transfected and non-transfected groups. The morphology of cardiomyocytes in the non-transfected group changed explicitly, but the change was not so obvious in the transfected cardiomyocytes. The expression of beta-tubulin and troponin increased significantly in the transfected group as compared with the non-transfected group. In the non-transfected group, numbers of apoptotic cardiomyocytes were significantly higher than in the transfected group. The c-jun protein, PKCa, and JNK were significantly expressed in the non-transfected group, and they reached a maximum at the 24th hour after cardiomyocytes were treated with burn serum and hypoxia. In the transfected group, however, expressions of c-jun protein, PKCa, and JNK decreased significantly compared with the non-transfected group. The c-jun antisense gene recombinant transfection alleviates injury to cardiomyocytes treated with burn serum and hypoxia, probably through low expression of PKCa and JNK.

Animals↗
PubMed1997

Regulation of the transfected Na+/H+-exchanger NHE3 in MDCK cells by vasotocin.

NHE3 is most likely the isoform involved in renal reabsorption of HCO3- and Na+. The functional properties of the "cloned" NHE3 isoform, including its transport regulation by extra- and intracellular stimuli, have so far been studied using non-epithelial expression systems. In the present report we stably transfected NHE3 cDNA (rabbit isoform) into Madin-Darby canine kidney cells (MDCK) cells and compared the sensitivity to inhibitors and the regulation of the Na+/H+-exchanger by vasotocin in NHE3 transfectants to that of the intrinsic basolateral Na+/H+-exchanger in untransfected and control transfected MDCK cells. By Southern blot analysis we documented that the NHE3 transcript is expressed in NHE3 transfectants. Na+/H+-exchange activity, measured as sodium-dependent recovery of intracellular pH from an acid load using 2', 7'-bis(carboxymethyl)-5(6)-carboxy-fluorescein (BCECF), was equally present at the basolateral cell surface of all cell lines; however, NHE3 transfectants demonstrated transport activity in the apical membrane that was significantly higher than that in untransfected or control transfected MDCK cells. Studies with ethylisopropylamiloride (EIPA) have shown that there is a similar sensitivity to inhibitors of the apical and/or basolateral Na+/H+-exchanger in transfected and untransfected MDCK cell lines. In contrast, the apical Na+/H+-exchanger (as compared to the basolateral Na+/H+-exchanger) of NHE3 transfectants was found to be relatively insensitive to the inhibitor HOE 694. Vasotocin decreased the activity of the apical Na+/H+-exchanger in NHE3 transfectants and stimulated the activity of the basolateral Na+/H+-exchanger in transfected (with NHE3 or pMAMneo) and untransfected MDCK cells. Phorbol ester, as expected, increased the activity of the Na+/H+-exchanger in the basolateral membrane of all cell lines; also, it stimulated transport activity at the apical cell surface of NHE3 transfectants. No change of Na+/H+-exchange activities was seen in studies with 8-bromo-cAMP. The PKC inhibitor calphostin C completely suppressed regulation of the apical and/or basolateral Na+/H+-exchanger by vasotocin, it partially blocked activation of the apical Na+/H+-exchanger in NHE3 transfectants by phorbol 12-myristate 13-acetate (PMA), and completely blocked stimulation of basolateral Na+/H+-exchanger by PMA. Consistent with a V1 receptor action, the effects of vasotocin in NHE3 transfectants and in MDCK cells were blocked by the V1 receptor antagonist, d(CH2)5Tyr(Me)-AVP, but were not reproduced by the V2 receptor agonist desmopressin. It is concluded that NHE3 in the apical membrane of NHE3-transfected MDCK cells contributes to the differential regulation of the apical and basolateral Na+/H+-exchanger by vasotocin; NHE3 is inhibited and endogenous Na+/H+-exchange activity is stimulated by vasotocin via V1 receptor activation of the protein kinase C pathway.

Animals↗
PubMed2005

Freeze-drying of cationic polymer DNA complexes enables their long-term storage and reverse transfection of post-mitotic cells.

WERI-Rb1 retinoblastoma (Rb) cell line, a human photoreceptor model, is notoriously difficult to transfect. Culturing of the WERI-Rb1 cells as a monolayer is complicated and cells are easily detached during transfection. Furthermore, transfection efficiencies in monolayer and in suspension are moderate at best which has limited the analysis of photoreceptor-specific promoters with low activity. To overcome these limitations, we developed a straightforward reverse transfection method for WERI-Rb1 cells wherein snap-frozen DNA/polyethylenimine complexes are freeze-dried on the surface of 48-well plates and stored in desiccator until cells are seeded for transfection. Comparing to conventional transfection, reverse transfection turned out to have equal or better transfection efficiency. In addition, while conventional transfection with cationic polymers requires serum-free conditions, reverse transfection can be performed in the presence of serum. Importantly, DNA/polyethylenimine complexes promote cell adhesion to the plates. This enables cell culturing as monolayers with concurrent complex uptake. Also, long-term storage of the plates did not reduce the transfection efficiency nor it had any effects on the cell toxicity. Because of the stability of complexes, reverse transfection enables large-scale transfection of hard-to-transfect retinoblastoma cells thus providing a reproducible, cost-effective and versatile tool for parallel screening of proteins and gene regulatory elements used in diverse applications.

Animals↗
PubMed2007

Optimization of transfection mediated by calcium phosphate for plasmid rAAV-LacZ (recombinant adeno-associated virus-beta-galactosidase reporter gene) production in suspension-cultured HEK-293 (human embryonic kidney 293) cells.

rAAV (recombinant adeno-associated virus) has become a very useful gene-delivery vector for gene therapy. However, it is very difficult to generate rAAV using triple transfection on a commercial scale, owing to its low productivity and inconveniently adhesive nature of its culture. An optimal suspension-culture transfection procedure was developed for rAAV-LacZ production in suspended HEK-293 cells mediated by calcium phosphate (lacZ, a reporter gene, codes for beta-galactosidase). The study showed that cytotoxicity of transfection complexes and cell aggregation in suspension culture were two key factors affecting high suspension-culture transfection efficiency. Cytotoxicity of transfection complexes was influenced effectively by mixture of Ca(2+) and plasmid DNA when their concentrations were decreased from 300 to 150 mM and from 3.0 to 1.5 microg/ml respectively, as manifested by a relatively higher cell viability after suspension-culture transfection. Moreover, the transfection efficiency was still less than 15%. In addition, we explored the disruption of cell aggregation and the control of transfection-complex size with 2.0 mM EGTA treatment for 30 min before transfection and the addition of 100 mM Mg(2+) during transfection respectively, procedures which enhanced transfection efficiency significantly, owing to more contact and endocytosis between cells and transfection complexes. Finally, the high transfection level and rAAV-LacZ titre achieved under optimized suspension-culture transfection conditions, namely 40% and 5 x 10(11) v.g. (vector genomes)/60 ml of medium respectively, is promising for the technique's application in the large-scale production of rAAV.

Adenoviridae↗