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PubMed1979

The effect of factors causing induction of DNA breaks on transfection of chicken cells by the XC DNA, and the kinetics of appearance of virus-producing cells after transfection.

The effect of BUdR and 4NQO treatment of the recipient chicken fibroblast cultures on the efficiency of transfection by the XC DNA was investigated. The efficiency of transfection was 2-fold higher when the recipient cultures were incubated in the presence of 10 micrograms BUdR/ml in medium 199 (48%) than when cultures were not treated (25%). The efficiency was not further increased by exposure of BUdR-treated cultures to visible light for 20 min (31%). Growth of BUdR-treated cultures decreased after light irradiation which indicated that BUdR was incorporated into the host cell DNA. Treatment of the recipient cultures with 4NGO at a concentration of 0.4 micrograms/ml for 2 h before transfection exerted only unfavourable effects on transfection efficiency. In cultures positive in transfection, transformation was first detected 3 days after exposure to DNA by the infectious centre assay, with a frequency of 4.76 +/- 6.46 transformed cells per 10(6) cells.

4-Nitroquinoline-1-oxide↗
PubMed1995

Characterization and epitope mapping of four HLA class II reactive mouse monoclonal antibodies using transfected L cells and human cells transfected with mutants of DQB1*0302.

To study epitopes of HLA class II molecules, four mouse monoclonal antibodies (mAbs) 13B6, 17F8, 19A1 and 12G6 were made using HLA-DQ8, DP2 and DP4 expressing mouse transfectants for immunization. Three of the mAbs, 13B6, 17F8 and 19A1, bound to all DQ1, 4, 8 or 9 positive B-lymphoblastoid cell lines (B-LCLs) and transfectants tested, i.e. cells carrying the DQB1 genes 0302-3, 0401-2, 0501-3, 0601-4 and 0609 irrespective of the accompanying DQA1 gene. These DQB1 genes code for the shared amino acids (aa) GVY in position 45-47 of the DQ beta chain. DQ1+4+8+9 specific (IIB3) and DQ3 specific (IVD12) reference mAbs inhibited binding of all three mAbs. Testing 13B6, 17F8 and 19A1 with cells made using aa substitutions in various positions of DQ beta 1*0302 indicated involvement of aa 45 in the epitopes of all three mAbs. The last mAb (12G6) bound to all B-LCLs and all DP transfected cells. However, only some DR transfectants and a single DQ transfectant (carrying DQA1*0201 and DQB1*0202) bound mAb 12G6. This reactivity pattern correlates with a shared sequence of aa (RFDSDVGE) in position 39-46 of DR- and DQ- and 37-44 of DP beta chains.

Amino Acid Sequence↗
PubMed1994

In vitro and in vivo growth of B16F10 melanoma cells transfected with interleukin-4 cDNA and gene therapy with the transfectant.

In an attempt to develop the most effective cytokine gene therapy, we transfected mouse interleukin(IL)-2, mouse IL-4, and human IL-6 cDNAs into mouse melanoma cells, B16F10. Transfection with IL-4 cDNA decreased the tumorigenicity of B16F10 most strongly. We investigated whether gene therapy with IL-4-transfected B16F10 cells was possible. Flow-cytometric analysis showed that major histocompatibility complex class I and II expression in B16F10 and IL-4-cDNA-transfected B16F10 (B16F10-IL4) cells did not differ. Doubling times of B16F10 and B16F10-IL4 were 20.1 and 21.1 h respectively. The growth of B16F10 cells was retarded if C57BL/6 mice were inoculated with B16F10-IL4 at the contralateral sides. When 5 x 10(5) B16F10 cells were transplanted subcutaneously into the flanks of C57BL/6 mice, they all developed a tumor mass, whereas no tumor masses formed in those transplanted with B16F10-IL4 cells within 60 days. No nude, severe combined immunodeficient or beige mice were able to reject parental B16F10 or B16F10-IL4 cells, although, B16F10-IL4 tumor growth in all these immunodeficient mice was slower than that of B16F10. Therefore, we concluded that T and natural killer cells are necessary for rejection of B16F10-IL4 tumor cells.

Animals↗
PubMed2000

DNA transfection and transfected cell viability using amphipathic asymmetric dendrimers.

Amphipathic asymmetric dendrimers have been investigated for use in delivery of genes into cells, with the objective of optimising transfection efficiency and maintaining cell viability. We have synthesised amphipathic asymmetric dendrimers by solid phase methods. The ability of two of these to transfect BHK cells in culture with beta-galactosidase gene was determined by X-gal staining. Cell viability was measured by the MTT assay for BHK cells, and by spectroscopy for lysis of erythrocytes. Interactions between dendrimer and DNA were investigated by agarose gel electrophoresis. BHK cells were optimally transfected at 5:1 +/- charge ratio yielding 20% cells receiving at least one copy of the plasmid. Cell viability decreased when the dendrimer to DNA ratio exceeded 5:1. Raising the pH significantly affected the electrophoretic mobility of complexes of dendrimer and DNA. We conclude that amphipathic asymmetric dendrimers enable efficient plasmid DNA uptake into BHK cells. Cell viability is maintained at high concentrations of dendrimer when complexed with DNA at a 5:1 +/- charge ratio. Efficiency of transfection and cell viability suggest the system may be suitable for gene delivery in vivo.

Animals↗
PubMed2001

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

Animals↗
PubMed2005

Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells.

An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation.

Animals↗
PubMed1973

Transfection of Escherichia coli spheroplasts. 3. Facilitation of transfection and stabilization of spheroplasts by different basic polymers.

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.

Coliphages↗
PubMed2003

Rescue of hepatitis A virus from cDNA-transfected but not virion RNA-transfected mouse Ltk- cells.

Hepatitis A virus (HAV) has stringent replication requirements and a restricted host-range. Mouse Ltk- cells do not support growth of HAV upon infection or transfection of virion RNA. However, low levels of HAV were rescued from Ltk- cells transiently transfected with its infectious cDNA. Ltk- stable transfectants that expressed HAV antigens and produced infectious HAV were selected and termed Ltk-pJH15 cells. After a few serial passages, HAV became undetectable in the Ltk-pJH15 cells. Multiple rounds of single cell cloning of HAV antigen positive Ltk-pJH15 cells resulted in the isolation of clone E8 that produced higher levels of HAV for at least 5 passages. HAV produced in E8 cells was similar to the parental virus as shown by infectivity assays. Luciferase assays using a bi-cistronic construct containing the HAV 5' noncoding region showed similar levels of HAV IRES-dependent translation in Ltk- and Ltk-pJH15 cells, which suggested that HAV IRES-dependent translation was not a limiting factor for HAV growth in these cells. The availability of the Ltk-pHJ15 cells will allow the identification of cellular factors required for HAV growth, which could lead to the development of a mouse model to study pathogenesis of HAV.

Animals↗
PubMed2004

On the disulfide-linker strategy for designing efficacious cationic transfection lipids: an unexpected transfection profile.

Herein, employing a previously reported disulfide-linker strategy, we have designed and synthesized a novel cationic lipid 2 with a disulfide-linker and its non-disulfide control analog lipid 1. The relative efficacies of lipids 1 and 2 in transfecting CHO, COS-1 and MCF-7 cells were measured using both reporter gene and whole cell histochemical staining assays. In stark contrast to the expectation based on the disulfide-linker strategy, the control non-disulfide cationic lipid 1 showed phenomenally superior in vitro transfection efficacies to its essentially transfection incompetent disulfide counterpart lipid 2. Results in DNase I protection experiments and the electrophoretic gel patterns in the presence of glutathione, taken together, are consistent with the notion that the success of the disulfide-linker strategy may depend more critically on the DNase I sensitivity of the lipoplexes than on the efficient DNA release induced by intracellular glutathione pool.

Animals↗
PubMed2004

New strategy for transfection: mixtures of medium-chain and long-chain cationic lipids synergistically enhance transfection.

To date, the primary approach to improve the transfection properties of cationic lipids has been the synthesis of new kinds of cationic amphipaths or the inclusion of noncationic helper lipids. Here, it is reported that an alternative approach can be unusually effective, namely, the combination of two cationic lipid derivatives having the same head group but tails of different chain lengths. Particularly efficient was the combination of dilauroyl (12 carbon chain) and dioleoyl (18 carbon chain) homologues of O-ethylphosphatidylcholine. This mixture transfected DNA into human umbilical artery endothelial cells (HUAEC) more than 30-fold more efficiently than either compound separately. A unique advantage of this kind of combination agent is that transfection can be optimized either in the presence or absence of serum by adjusting the component ratio.

Cations↗
PubMed1986

Differential responsiveness of myc- and ras-transfected cells to growth factors: selective stimulation of myc-transfected cells by epidermal growth factor.

To identify functional relationships between oncogenes and growth factors, we compared the effects of transfected myc and ras oncogenes on the responsiveness of Fischer rat 3T3 cells to three growth factors: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-beta). Control cells did not grow in soft agar under any conditions. ras-Transfected cells grew in soft agar under all conditions tested and were insensitive to the stimulatory effects of exogenous growth factors. These cells secreted elevated levels of both EGF-like factors and TGF-beta, suggesting that the lack of responsiveness of these cells to exogenous growth factors arose from autocrine stimulation. myc-Transfected cells displayed conditional anchorage-independent growth: they formed numerous colonies in soft agar in the presence of EGF but relatively few colonies in the presence of PDGF or TGF-beta. Secretion of EGF-like factors and TGF-beta by these cells was not elevated above that of control cells. These results suggest a model for the mechanism of cooperation between myc and ras oncogenes in which ras-like genes induce growth factor production, while myc-like genes increase the responsiveness of cells to these factors.

Animals↗
PubMed1992

Cytokinesis is more rapid in Ha-T24-ras transfected rat embryo fibroblasts than in non-transfected control cells.

It has long been known that neoplastic cells are characterized by increases in cell motility. Earlier studies from this laboratory indicated that mitotic events were also altered in many tumor and experimentally transformed cells and that this included increases in metaphase duration and a reduction in the duration of cytokinesis. The studies presented in this paper were done to determine whether or not transfection of normal rat embryo fibroblasts by the Ha-T24-ras oncogene could also produce such alterations in mitotic events. The results obtained with the use of time lapse video microscopy indicate that neither the duration of metaphase nor the rate of chromosome movement during anaphase was altered but that the rate of furrow progression during cytokinesis occurred at a significantly more rapid rate. Thus, the cellular alterations induced by transfection with Ha-T24-ras accelerate microfilament-dependent cytokinetic furrowing without significant effects on microtubule-dependent mitotic events. One of several possible mechanisms that could account for these observations involves a down regulation of protein kinase C which has been reported to occur in many neoplastic cells including those transformed by ras. Such a hypothesis could also have broader implications because it may be applicable to the increase in motility and metastatic activity generally observed in transformed cells.

Animals↗
PubMed2001

Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts.

The efficiency of nuclear transfer (NT) using two primary cultures of fetal fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence is placed under the control of the bovine alpha(S1)-casein promoter (TFF1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgenic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos reconstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage at a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were significantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear donors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NT embryos derived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majority of pregnancies (8/11; 73%) was lost in the first and second trimesters of gestation, whereas 4/11 (36%) pregnancies with FF2 embryos were lost during the full period of in vivo development. Of 11 FF2 and 6 TFF2 born calves (25 and 13% of transferred embryos, respectively), 6 and 3 survived including one oversized FF2 calf. After transfer of TRFF1 and TRCF1 NT embryos to recipients, initial pregnancy rate was as a tendency higher in the TRFF1 (49%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancies and all TRCF1 pregnancies were lost in the first and second trimester. A high proportion of TRFF1 calves (5/8) showed increased body weights, and only two calves which were also large survived. These findings demonstrate that (i) extended culture associated with transfection and selection procedures may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning.

Animals↗
PubMedDate not supplied

Combined I-124 positron emission tomography/computed tomography imaging of NIS gene expression in animal models of stably transfected and intravenously transfected tumor.

PURPOSE: With the advent of replication competent viruses for cancer gene therapy, it has become imperative to monitor the biodistribution, expression and replication of these vectors in living organisms. We evaluated the potential of I-124 positron emission tomography (PET)/computed tomography (CT) imaging in gene therapy animal models utilizing the sodium iodide symporter (NIS) and compared the findings to I-123 gamma camera imaging. PROCEDURES: CB17 SCID mice were implanted with myeloma cell lines expressing NIS or infected by MV-NIS given systemically. Mice were imaged by both gamma camera (I-123) and PET/CT (I-124 ) and image quality assessed. RESULTS: NIS expressing tumors concentrated 7.1% of the injected activity while tumors infected with the control virus had only 0.3% of the activity injected. CONCLUSIONS: I-124 PET/CT in combination with NIS allows the tracking of stably transfected tumors or intravenously transfected tumors. Combined modality imaging using PET/CT allows accurate and non-invasive imaging of the distribution and gene expression of a replicating viral vector in living systems.

Animals↗
PubMed1988

Analysis of cellular expression of gangliosides by gene transfection. I: GD3 expression in myc-transfected and transformed 3Y1 correlates with anchorage-independent growth activity.

Transfection of c-myc DNA into rat fibroblastic 3Y1 cell line resulted in the neosynthesis of GD3 ganglioside, as has been previously shown to occur after transfection of 3Y1 cells with adeno El (Nakakuma, H., Sanai, Y., Shiroki, K., and Nagai, Y. (1984) J. Biochem. 96, 1471-1480); in both cases, the products are expressed intranuclearly. Moreover, a clear correlation was identified between levels of GD3 expression and colony-forming activity (in soft agar) of myc-transformed 3Y1 cells, implying that GD3 plays some specific role in myc-induced transformation of 3Y1 cell line.

Animals↗
PubMed2005

Effect of pesticides on estrogen receptor transactivation in vitro: a comparison of stable transfected MVLN and transient transfected MCF-7 cells.

The estrogenic potential of four pesticides (endosulfan, prochloraz, tolchlofos-methyl and propamocarb) was compared in parallel with 17beta-estradiol (E2) by reporter constructs in transient transfected MCF-7BUS and in stable transfected MVLN cells. Similar detection limit and half maximum effect concentration was determined for E2, whereas the maximum effect concentration of E2 was much higher in MCF-7BUS (10 nM) than in MVLN (150 pM), with the induced response being approximately six times the level in MVLN cells. Alone the four pesticides elicited the same relative response in the two bioassays, and similar data was obtained upon co-exposure with E2 for endosulfan and propamocarb. In contrast to the transient MCF-7BUS system, endosulfan further increased the E2 induced response in MVLN cells, whereas propamocarb did not induce the E2 response in MVLN cells as observed in MCF-7BUS cells. In conclusion, high agreement between the two reporter assays was observed, although some performance characteristics have to be considered.

Biological Assay↗
PubMed1998

Tumor growth of FGF or VEGF transfected MCF-7 breast carcinoma cells correlates with density of specific microvessels independent of the transfected angiogenic factor.

We have previously shown that fibroblast growth factor (FGF)-1-, FGF-4-, or vascular endothelial growth factor (VEGF/VPF)-transfected MCF-7 breast carcinoma cells growing as tumors in nude mice are tamoxifen resistant and/or estrogen independent. These transfectants provide opportunity for study of in situ tumor-induced angiogenesis promoted by the individual angiogenic factors under growth-promoting versus growth-inhibiting hormonal conditions. In the present study, vessels in tumors harvested at varying times after tumor cell injection were immunohistochemically highlighted and vessel morphology and topography were scored on a scale of 0 to 4 by blinded observers. In tumors produced by all cell lines under all growth-promoting hormonal conditions, there was significantly increased abundance (P < 0.05) of edge-associated and intratumor microvessels, but not of stromally located microvessels, when compared with tumor nodules harvested under growth-inhibiting conditions, regardless of the identity of the angiogenic factor or the hormonal treatment. Image analysis of bromodeoxyuridine (BrdU)-labeled nuclei of tumors produced by all cell lines under all hormonal conditions harvested at early time points showed that mean labeling indices were highest for hormonal conditions that produced the most robust growth in that particular cell line, implying that a high BrdU labeling index is a predictor of future tumor growth in individual tumors. These results confirm previous studies that established the importance of neovascularization for tumor growth and provide validation for use of these cell lines to study the process of angiogenesis in vivo. Study of gene expression in endothelial cells in edge-associated or intratumor vessels using this model might reveal mechanisms important in tumor-induced angiogenesis in human breast cancer.

Animals↗