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Biomedical subjects

N S Yang

Publications and source records attributed to N S Yang.

At least 19 recordsLinked to original sources
PubMed2001

Antioxidant activity of extracts from Acacia confusa bark and heartwood.

The antioxidant activity of extracts from bark and heartwood of Acacia confusa was evaluated by various antioxidant assays, including free radical and superoxide radical scavenging assays and lipid peroxidation assay as well as hydroxyl radical-induced DNA strand scission assay. In addition, an ex vivo antioxidant assay using a flow cytometric technique was also employed in this study. The results indicate that both bark and heartwood extracts clearly have strong antioxidant effects. Similar inhibitory activities for each test sample were found for both 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical generation and lipid peroxidation. As for the superoxide radical scavenging activity, the heartwood extract was more effective than the bark extract. Furthermore, the heartwood extract protected PhiX174 supercoiled DNA against strand scission induced by ultraviolet photolysis of H2O2, and it reduced the amounts of intracellular hydrogen peroxide, a reactive oxygen species, when it was co-incubated with human promyelocytic leukemia (HL-60) cells under oxidative stress.

Acacia↗
PubMed2001

Effective particle-mediated vaccination against mouse melanoma by coadministration of plasmid DNA encoding Gp100 and granulocyte-macrophage colony-stimulating factor.

Particle-mediated gene delivery was used to immunize mice against melanoma. Mice were immunized with a plasmid cDNA coding for the human melanoma-associated antigen, gp100. Murine B16 melanoma, stably transfected with human gp100 expression plasmid, was used as a tumor model. Particle-mediated delivery of gp100 plasmid into the skin of naïve mice resulted in significant protection from a subsequent tumor challenge. Co-delivery of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) expression plasmid together with the gp100 plasmid consistently resulted in a greater level of protection from tumor challenge. The inclusion of the GM-CSF plasmid with the gp100 DNA vaccine allowed a reduction in the gp100 plasmid dose required for antitumor efficacy. Protection from tumor challenge was achieved with as little as 62.5 ng of gp100 DNA per vaccination. Tumor protection induced by the gp100 + GM-CSF gene combination was T cell mediated, because it was abrogated in vaccinated mice treated with anti-CD4 and anti-CD8 monoclonal antibodies. In addition, administration of the gp100 + GM-CSF DNA vaccine to mice bearing established 7-day tumors resulted in significant suppression of tumor growth. These results indicate that inclusion of GM-CSF DNA augments the efficacy of particle-mediated vaccination with gp100 DNA, and this form of combined gp100 + GM-CSF DNA vaccine warrants clinical evaluation in melanoma patients.

Animals↗
PubMed2000

Interleukin-12 gene therapy of a weakly immunogenic mouse mammary carcinoma results in reduction of spontaneous lung metastases via a T-cell-independent mechanism.

In our previous studies using gene gun-mediated delivery of interleukin 12 (IL-12) cDNA in vivo, we observed T-cell-mediated regression of established murine tumors and demonstrated the induction of systemic immunity in test animals. In this study, we further characterized the antitumoral and anti-metastatic effect of this gene therapy approach by employing two murine metastatic mammary tumor models: the immunogenic TS/A adenocarcinoma and the weakly immunogenic 4T1 adenocarcinoma. In the TS/A model, gene transfer into the skin overlying an established intradermal tumor with an IL-12 cDNA expression vector resulted in complete tumor regression in 50% of mice followed by the development of immunological memory. In contrast, the growth of the intradermal 4T1 tumors was not affected by the IL-12 gene therapy protocol. However, this treatment resulted in a substantial reduction of spontaneous metastases in the lungs of 4T1 tumor-bearing mice and significantly prolonged their survival time. T cells were not required for this anti-metastatic effect, because it was also observed in nude mice and in mice depleted of CD4+ and CD8+ T cells. Tumor-draining lymph node cells obtained from 4T1 tumor-bearing mice treated with IL-12 cDNA exhibited increased natural killer (NK) activity and produced enhanced levels of interferon-gamma (IFN-gamma) compared with similar mice treated with luciferase cDNA. In addition, in vivo depletion of NK cells or neutralization of IFN-gamma resulted in partial suppression of the anti-metastatic effect of IL-12 gene therapy, suggesting the involvement of both NK cells and IFN-gamma in this effect.

Adenocarcinoma↗
PubMed2000

Regulation of cell proliferation, gene expression, production of cytokines, and cell cycle progression in primary human T lymphocytes by piperlactam S isolated from Piper kadsura.

Effects of piperlactam S (C(17)H(13)NO(4); mol. wt. 295) isolated from Piper kadsura on phytohemagglutinin (PHA) stimulated cell proliferation were studied in primary culture of human T cells. The results showed that piperlactam S suppressed T cell proliferation at about 0 to 12 h after stimulation with PHA. Synthesis of total cellular proteins and RNA in activated cell cultures was also suppressed. The inhibitory action of piperlactam S was not through direct cytotoxicity. Cell cycle analysis indicated that piperlactam S arrested the cell cycle progression of activated T cells from the G(1) transition to the S phase. In an attempt to further localize the point in the cell cycle at which arrest occurred, a set of key regulatory events leading to the G(1)/S boundary, including gene expression of cytokines and c-Fos protein synthesis, was examined. Piperlactam S suppressed, in activated T lymphocytes, the production and mRNA expression of cytokines such as interleukin-2 (IL-2), IL-4, and interferon-gamma in a dose-dependent manner. In addition, Western blot analysis indicated that c-Fos protein expressed in activated T lymphocytes was decreased by piperlactam S. Results of kinetic study indicated that inhibitory effects of piperlactam S on IL-2 mRNA expressed in T cells might be related to blocking c-Fos protein synthesis. Thus, the suppressant effects of piperlactam S on proliferation of T cells activated by PHA seemed to be mediated, at least in part, through inhibition of early transcripts of T cells, especially those of important cytokines, IL-2, IL-4, and arresting cell cycle progression in the cells.

Adult↗
PubMed1999

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1beta converting enzyme cDNA.

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1beta converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-gamma. First, we compared the levels of IFN-gamma secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-gamma production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-gamma levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8(+) T cell-mediated and partially IFN-gamma-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8(+) T cells and IFN-gamma-dependent pathways.

Adenocarcinoma↗
PubMed1999

Gene gun-mediated IL-12 gene therapy induces antitumor effects in the absence of toxicity: a direct comparison with systemic IL-12 protein therapy.

Using three murine tumor models, we compared the antitumor efficacy and certain physiological effects of an in vivo interleukin-12 (IL-12) gene therapy protocol and a systemic IL-12 protein therapy protocol. An IL-12 cDNA gene construct was administered in situ into skin tissue via gene gun delivery, and recombinant IL-12 protein was administered subcutaneously at a dose of 1 microgram/mouse/treatment. Both treatment regimes induced a comparable level of regression of established intradermal MethA sarcomas. In B16 melanoma and P815 mastocytoma models, antitumor efficacy of IL-12 protein therapy appeared to be slightly higher than that of IL-12 gene therapy; however, the protein therapy protocol in this comparative study resulted in a high level of mortality of mice. It was also demonstrated that IL-12 gene therapy, in contrast to the IL-12 protein therapy, was not associated with weight loss, splenomegaly, increased Ly6 antigen expression in the spleen, or visible signs of toxicity, such as fur ruffling and lethargy. Moreover, serum levels of interferon-gamma (IFN-gamma) induced in response to IL-12 gene therapy were 300-1000 times lower than those induced by the systemic IL-12 protein administration. Together, these results suggest that gene gunmediated in vivo delivery of IL-12 cDNA may be considered as a safer alternative to IL-12 protein therapy for certain human cancers.

Animals↗
PubMed1998

Cytokine transgene expression and promoter usage in primary CD34+ cells using particle-mediated gene delivery.

Induction or short-term transgenic expression of specific cytokines, growth factors, or other candidate therapeutic genes in hematopoietic progenitor or stem cells is potentially applicable to gene therapy for cancer. In this study, we explored the application of a gene gun technique, as an alternative to viral vectors, for ex vivo gene transfer into and transient gene expression in highly enriched CD34+ cells derived from human umbilical cord blood. Twenty-four hours posttransfection, 32.6 to 1500 pg/l x 10(6) CD34+ cells of transient gene expression was routinely obtained for specific cytokine and reporter genes. Transgene expression at the single-cell level was revealed by X-Gal staining of lacZ cDNA-transfected CD34+ cells. Expression of four candidate therapeutic genes, namely human granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, interleukin 2, and interferon gamma, was detectable for 4 to 7 days in CD34+ cells. A human elongation factor 1alpha promoter/intron 1 transcription unit was identified as a strong cellular promoter for CD34+ cells, exhibiting strength similar to that of the commonly employed cytomegalovirus immediate-early promoter. These results suggest that the nonviral, gene gun technique offers an efficient alternative approach for transient transgenic studies of hematopoietic cells and may provide new possibilities for certain cancer gene therapy strategies using CD34+ cells.

Antigens, CD34↗
PubMed1998

Development of human granulocyte-macrophage colony-stimulating factor-transfected tumor cell vaccines for the treatment of spontaneous canine cancer.

Cytokine gene-engineered tumor vaccines are currently an area of intense investigation in both basic research and clinical medicine. Our efforts to utilize tumor vaccines in an immunotherapeutic manner involve canines with spontaneous tumors. We hypothesized that canine tumor cells, transfected with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) cDNA in a plasmid vector, would prove nontoxic following intradermal administration, generate biologically relevant levels of protein, effect local histological changes at the sites of vaccination, and create a systemic antitumor response. Sixteen tumor-bearing dogs were admitted to a study of ex vivo gene therapy. Tumor tissue was surgically removed, enzymatically and mechanically dissociated, irradiated, transfected, and intradermally injected back into the patients. The dogs were vaccinated with primary autologous tumor cells transfected with hGM-CSF or a reporter control gene. hGM-CSF protein was detected (0.07 to 14.15 ng/vaccination site) at 24 hr postinjection and dramatic histological changes were observed, characterized by neutrophil and macrophage infiltration at the sites of injection of hGM-CSF-transfected tumor cells. This was in stark contrast to the lesser neutrophilic and eosinophilic infiltrates found at control vaccination sites. Objective evidence of an antitumor response was observed in three animals. These data, in a large animal translational model of spontaneous tumors, demonstrate in vivo biological activity of hGM-CSF-transfected autologous tumor cell vaccines.

Animals↗
PubMed1998

Broadened clinical utility of gene gun-mediated, granulocyte-macrophage colony-stimulating factor cDNA-based tumor cell vaccines as demonstrated with a mouse myeloma model.

Effective immunization against the murine B16 melanoma by a nonviral approach in which a gene gun is used to transfer GM-CSF cDNA into tumor cells has been described. We have extended this nonviral approach by using the poorly immunogenic murine myeloma MPC11 model. Vaccination with the transfected, GM-CSF-expressing MPC11 cells induced a potent antitumor cytotoxic T lymphocyte response associated with tumor rejection in the majority of the test mice. Furthermore, nearly 100% (27 of 28) of the tumor-free mice were able to reject a tumor rechallenge. While this approach is clinically attractive because of minimal tissue manipulation/culturing and the absence of infectious agents, a number of tested human primary tumors, including myeloma cells, have failed to produce high levels of GM-CSF after gene gun transfection. To circumvent the low transfection efficiency in certain human tumor cells, we showed that combining irradiated tumor cells to provide tumor antigens together with gene gun-transfected fibroblasts to provide GM-CSF induced effective tumor rejection. We also report that normal human skin fibroblasts transfected by the gene gun produce high levels of human GM-CSF (250 ng/10(6) cells/24 hr). These results suggest that combining irradiated tumor cells with gene gun-transfected fibroblasts results in antitumor immune responses and may allow for a wider application of this approach to cancer immunotherapy.

3T3 Cells↗
PubMed1997

Cytokine gene therapy of cancer using gene gun technology: superior antitumor activity of interleukin-12.

We compared the antitumor effect of several transgene expression plasmids encoding specific cytokines, including interleukin-2 (IL-2), IL-4, IL-6, IL-12, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF), following gene gun-mediated DNA delivery into the epidermis overlying an established intradermal murine tumor. IL-12 gene therapy was much more effective than treatment with any other tested cytokine gene for induction of tumor regression. Strong activation of antitumor immunity in response to IL-12 gene therapy was evidenced by an augmented CD8+ T cell-mediated cytolytic activity in the draining lymph nodes of tumor-bearing mice. Furthermore, following the IL-12 gene therapy protocol, test mice were able to eradicate not only the treated but also the untreated solid tumors at distant sites. This systemic antitumor effect of IL-12 gene therapy was not associated with visible signs of toxicity or significantly elevated systemic levels of IFN-gamma. These results show that gene gun-mediated in vivo delivery of IL-12 cDNA clearly distinguishes itself from the other cytokine gene therapy approaches tested in parallel, suggesting that this delivery system may be employed as an efficient model for comparative studies of in vivo cytokine gene therapy. The results also suggest that the current IL-12 gene therapy strategy may provide a safer alternative to IL-12 protein therapy for clinical treatment of cancers.

Animals↗
PubMed1997

Phase I/IB study of immunization with autologous tumor cells transfected with the GM-CSF gene by particle-mediated transfer in patients with melanoma or sarcoma.

The objective of this Phase I study is to assess the acute and long-term toxicities of intradermal vaccination of cancer patients with lethally-irradiated tumor cells that have been transfected by particle-mediated gene transfer (PMGT) with gold particles coated with human granulocyte-macrophage colony stimulating factor (GM-CSF) DNA in a plasmid expression vector. The GM-CSF DNA-coated gold particles are delivered to tumor cells using helium pressure with a hand held gene delivery device. Preclinical studies have demonstrated that vaccination of mice with irradiated, GM-CSF-transfected melanoma cells provided protection from subsequent challenges with non-irradiated, non-transfected tumor cells. Ongoing human tumor immunotherapy studies use patients' melanoma or renal carcinoma cells transfected with a retroviral vector containing GM-CSF cDNA as a vaccine to elicit anti-tumor immune responses. PMGT transfection, unlike retroviral transfection, does not require tumor cells to proliferate in vitro to undergo gene transfer. Instead, tumor tissue can be dissociated into small tissue clumps or cell aggregates and then immediately transfected using the gene gun. PMGT physically inserts the DNA without the need for cell surface interaction with viral components or exposure of the patient to viral antigens. As described in this protocol, fresh human sarcoma and melanoma specimens can be transfected with the GM-CSF DNA-coated gold particles with subsequent production of biologically active GM-CSF protein. In this study tumor tissue will be obtained from patients with melanoma or sarcoma. Tumor tissue will be dissociated, irradiated, and transfected with GM-CSF DNA by PMGT. In this ascending dosage study, two dose levels of GM-CSF DNA will be studied in 2 groups of 6 patients each. Patients will receive two intradermal injections of the irradiated, transfected tumor in a single extremity. On days 3 and 14 post-vaccination, patients will undergo surgical excision of the vaccination sites to assess GM-CSF production and infiltration of immune effector cells. On Day 25, patients will undergo DTH testing with intradermal injection in their opposite extremity of 5 x 10(6) irradiated non-transfected autologous tumor cells cryopreserved at the time of vaccine preparation. This injection site will be assessed on day 28 post-vaccination and surgical excision of the DTH testing site will be performed on day 28 if a positive reaction is noted. The patients will be observed for local and systemic toxicity on days 2, 3, 5, 8, 14, 25, and 28 after the vaccination. Restaging of the patients' disease and long term toxicity evaluation will be performed at 3, 6, and 12 months and then yearly.

Animals↗
PubMed1997

DNA cancer vaccines: a gene gun approach.

A wide variety of approaches, all using gene transfer, have been tested experimentally as alternative means to vaccinate against cancer, either prophylactically or therapeutically. These include both ex vivo and in vivo gene transfer to tumour and/or non-tumour cells, using both viral and non-viral vectors. The transferred DNA has varied widely as well, including genomic or cDNA encoding tumour-associated or oncofoetal antigens, cytokines, histocompatibility molecules, and costimulatory molecules. Several of these approaches have been applied in human clinical trials. This review summarizes those approaches, then compares and evaluates various methods using cytokine DNA in conjunction with autologous tumour cells, with particular emphasis on particle-mediated gene transfer via a gene gun. Finally, prospects and needs for further development are discussed.

Animals↗
PubMed1996

Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine.

The necessity for prolonged tissue culture manipulations limits the clinical application of many form of gene therapy in patients with malignancies. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a plasmid expression vector could be effectively introduced into resting tumor cells, without the need for tissue culture propagation prior to or following transfection, and that efficient expression of transgenic GM-CSF by the transfected tumor cells would confer an effective immune response against tumors. GM-CSF cDNA in expression vectors was coated onto gold particles and accelerated with a gene gun device into mouse and human tumor cells. Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice. Transfected and irradiated murine B16 melanoma cells produced > or = 100 ng/ml murine GM-CSF/10(6) cells per 24 hr in vitro for at least 10 days. The antitumor efficacy of this nonviral approach was tested using irradiated B16 tumor cells that were transfected with mGM-CSF cDNA and injected into mice as tumor "vaccine". Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals wer protected from the tumor by the prior vaccine treatment. In contrast, only 2% of control animals were protected by prior treatment with irradiated B16 cells transfected with the vector containing the luciferase gene. These results suggest that particle-mediated transfection of fresh tumor explants with cytokine cDNA is an effective and clinically attractive approach for cancer therapy.

Animals↗
PubMed1996

Injection of complementary DNA encoding interleukin-12 inhibits tumor establishment at a distant site in a murine renal carcinoma model.

Interleukin (IL-12) protein has been shown to elicit diverse immunological responses and potent antitumor activity. We demonstrate here that intradermal injection of IL-12 cDNA induces systemic biological effects characteristic of the cytokine in vivo. Intradermal injection of IL-12 cDNA resulted in local expression of IL-12 mRNA, which correlated with a 10-fold increase in natural killer activity and a 3-4-fold increase in anti-CD3-induced IFN-gamma production in cultured splenocytes. Furthermore, when challenged with Renca tumor cells at a distant site, the day of tumor emergence was significantly delayed, and tumor growth was reduced in mice that received IL-12 cDNA, compared to mice given injections of plasmid vector alone. A number of the mice receiving IL-12 cDNA injections remained tumor free months after tumor challenge. In contrast to mice receiving recombinant IL-12 protein, no splenomegaly was detected when natural killer activity was significantly induced in mice receiving injections of IL-12 cDNA. Because purified plasmid DNA is more economical to prepare and has a longer shelf-life than recombinant proteins, and intradermal administration of cDNA encoding IL-12 did not cause splenomegaly, our findings suggest that the in vivo injection of cDNA encoding IL-12 may be a less toxic and more cost-effective alternative to IL-12 protein therapy in some clinical or experimental therapeutic applications.

Animals↗
PubMed1996

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

Animals↗
PubMed1996

CWR22 xenograft as an ex vivo human tumor model for prostate cancer gene therapy.

BACKGROUND: Lack of well-defined relevant in vivo or in vitro tumor models is one of the major limitations in assessing candidate therapeutic regimens, especially gene therapy, for prostate cancer. Since gene therapy is emerging as a potentially powerful therapeutic modality, it is desirable to evaluate this approach for the treatment of human prostate cancer. PURPOSE: We sought to establish a relevant ex vivo tumor model for gene therapy studies of human prostate cancer. METHODS: We constructed and established a transgenic human tumor model consisting of three major components: 1) human primary prostate cancer cells, CWR22, reactivated for growth after storage in liquid nitrogen; 2) a collagen gel ex vivo tissue culture system useful for short-term maintenance and manipulation of CWR22 cells under in vitro experimental conditions; and 3) a high-velocity, particle-mediated gene transfer system that is highly efficient in the ex vivo transfection of target cells. Prostate-specific antigen (PSA) levels in the cell culture media were monitored after transfecting CWR22 cells with candidate therapeutic genes, including the cytokines human interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), both as complementary DNAs [cDNAs]). CWR22 cells, transfected with firefly luciferase cDNA as a reporter gene, served as control cells for cytokine gene expression. CWR22 cells, transfected with the bacterial beta-galactosidase cDNA as a reporter gene, were used to assess the efficiency of gene transfer. Transcription of each of the cDNAs was driven by the cytomegalovirus (CMV) early gene promoter. RESULTS: The three-dimensional organization of tumor cells and functional characteristics of human prostate cancers were maintained in this ex vivo model of prostate cancer. Candidate therapeutic genes, CMV-IL-2 and CMV-GM-CSF, were expressed at peak levels of up to 38 ng of protein per 10(6) cells every 24 hours. IL-2 and GM-CSF secretion was sustained at approximately 40%-50% of peak levels during the entire experimental period (9-10 days in culture). At 7 days after gene delivery, a more than twofold reduction in the secretion of PSA was detected in the IL-2 (3.8 +/- 1.3 ng/10(4) cells every 24 hours [mean +/- standard deviation]) or GM-CSF (4.0 +/- 1.7 ng/10(4) cells every 24 hours) cDNA transfected cells as compared with the control cells transfected with luciferase cDNA (9.3 +/- 1.0 ng/10(4) cells every 24 hours). Up to 10% of the cells transfected with beta-galactosidase cDNA expressed measurable beta-galactosidase activity. CONCLUSION: This study demonstrated an efficient, rapid, and reliable system for gene transfer and expression in primary human prostatic carcinoma cells maintained in a collagen gel culture system. IMPLICATIONS: Our findings suggest a broad application of this CWR22 xenograft primary culture system as an ex vivo tumor model for the evaluation and characterization of various candidate therapeutic genes for human prostate cancer gene therapy, including a cytokine gene-modified tumor vaccine strategy.

Animals↗
PubMed1996

Efficient and sustained transgene expression in mature rat oligodendrocytes in primary culture.

In order to evaluate the characteristics and efficiency of gene transfer in primary cultures of oligodendrocytes, four different techniques including particle bombardment (Accell gene gun), cationic liposome-mediated transfection (lipofection), calcium phosphate co-precipitation and retroviral infection were compared using the LacZ and luciferase reporter genes. Highly purified postnatal adult rat oligodendrocytes were obtained by sequential immunopanning, plated in culture, and transfected using various reporter and promoter genes. The most efficient expression of LacZ and luciferase genes was found with particle mediated gene delivery. The transgene expression level obtained with gene gun delivery was at least two- to 100-fold greater than three other tested gene transfer methods. Comparison of the relative strength of four viral and two cellular promoters in these primary oligodendrocytes cultures demonstrated that the CMV promoter was the strongest. Using a human growth hormone (hGH) reporter gene, a long-term transgene expression pattern in primary oligodendrocytes was demonstrated to be sustained in culture for the entire experimental period (4 weeks) after particle-mediated gene transfer. These results demonstrate that expression of a foreign gene can be effectively achieved in primary cultures of adult oligodendrocytes, especially by using the particle bombardment method. The results also suggest that the current ex vivo gene transfer system may be used to manipulate oligodendrocytes for future application in gene therapy studies.

Adenoviridae↗