Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transfection”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10Linked to original sources
PubMed1981

Phi W-14 DNA inhibits transfection of Bacillus subtilis by SPP1 DNA.

The DNA of bacteriophage phi W-14 is unusual in that half of the thymine residues are replaced with the hypermodified pyrimidine alpha-putrescinylthymine (Kropinski et al., Biochemistry 12:151-157, 1973). Bacteriophage phi W-14 DNA and Bacillus subtilis DNA exhibited comparable competing abilities for the uptake of transfecting bacteriophage SPP1 DNA by competent cells of B. subtilis. B. subtilis DNA decreased transfection and uptake to the same extent, indicating that it merely competed with SPP1 DNA for uptake. Phi W-14 DNA, however, decreased transfection up to 30 times more effectively than it inhibited uptake. Phi W-14 DNA did not alter the kinetics of transfection. The degree of inhibition of transfection was dependent upon the time of addition of Phi W-14 DNA relative to the time of addition of SPP1 DNA. If failed to inhibit when added 30 min after SPP1 DNA. It had a fourfold-greater effect when added 10 min before, rather than simultaneously with, SPP1, but this enhancement was abolished by high concentrations of SPP1 DNA. The nature of the transfection process was not altered in those cells escaping inhibition by Phi W-14 DNA: two molecules of transfecting SPP1 DNA were required to form a transfectant with or without Phi W-14 DNA. Free putrescine did not affect transfection by SPP1 DNA. It was concluded that the putrescine groups covalently attached to phi W-14 DNA allowed this DNA to interfere with the transfection process at the intracellular level.

Bacillus subtilis↗
PubMed1982

Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency.

We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.

Animals↗
PubMed2001

K-Cl cotransport: immunohistochemical and ion flux studies in human embryonic kidney (HEK293) cells transfected with full-length and C-terminal-domain-truncated KCC1 cDNAs.

Coupled K and Cl movements are mediated by several isoforms of the K-Cl cotransporter (COT) encoded by the KCC genes. The ubiquitous KCC1 isoform, important for cell volume and ion homeostasis, has 12 transmembrane domains (Tmds), and cytoplasmic N- and C-terminal domains (Ntd and Ctd). This study investigates the cellular localization of KCC1 by confocal microscopy, activation of K-Cl COT by various non-osmotic and osmotic interventions with net unidirectional K and Rb fluxes at 37( degrees )C, and the effect of Ctd deletion on K-Cl COT regulation. Human embryonic kidney (HEK293) cells were transfected with full-length (fl) rabbit (rb)KCC1 and - CtdKCC1 cDNAs obtained after truncation at nucleotide 2011. Normal cells exposed to polyclonal anti-Ctd antibodies against Ctd epitopes within a 77 amino acid sequence (a.a.943-1020) revealed granular membrane and cytoplasmic immunostaining, presumably endogenous KCC1. Additional diffuse membrane and cytoplasmic immunofluorescence in flKCC1-transfected cells was absent in -CtdKCC1-transfected cells. Monoclonal antibodies against a c-myc epitope at the protein Ntd showed both membrane and cytosolic fluorescence. Basal and N-ethylmaleimide (NEM)-stimulated Rb influxes through K-Cl COT, calculated as Cl-dependent Rb fluxes, were 2-3-fold higher in flKCC1-transfected than in normal cells. NEM stimulation of K-Cl COT was highest in flKCC1-transfected cells, significantly lower in stably and abrogated in transiently -CtdKCC1-transfected cells. Furosemide, calyculin and genistein inhibited basal and NEM-stimulated K-Cl COT in normal and transfected cells. Staurosporine and hydroxylamine were ineffective stimulators. No effect of pH(0) changes (6.3-8.4) was observed in basal or NEM-stimulated K-Cl COT, in both normal and transfected cells. However, inhibition by NEM occurred at pH(0) 8.4. Furthermore, in a Cl-independent manner, NEM lowered cell K content by >30% and hypotonicity (210-70mOsM) stimulated furosemide-sensitive Rb influx and K loss. Thus, in cultured normal and KCC1-transfected cells, K-Cl COT shows significant differences from erythrocytes, and NEM and cell swelling open furosemide-sensitive and Cl-independent K/Rb channels. Failure of K-Cl COT in cells transfected with Ctd-truncated KCC1 to respond to NEM suggests a role of the Ctd for signal transduction.

Carrier Proteins↗
PubMed2005

Adhesion molecule expression in fibroblasts: alteration in fibroblast biology after transfection with LOX-1 plasmids.

The endothelial lectinlike, oxidatively (ox-) modified LDL receptor LOX-1 is a critical player in the pathogenesis of atherosclerosis and myocardial ischemia. Ox-LDL binding of LOX-1 results in the expression of various adhesion molecules, which attract monocytes to endothelial cells, an initial step in atherogenesis. We wished to examine the role of the ox-LDL/LOX-1 signaling pathway in fibroblasts, which naturally express low levels of LOX-1. Rat cardiac fibroblasts were transfected with either cytomegalovirus (CMV)-LOX-1wt (amino acids [aa] 1 to 273) or CMV-LOX-1(1-261) (an ox-LDL-binding negative mutant, aa 1 to 261) plasmid. Western blots showed that LOX-1 protein expression was increased significantly in cells transfected with CMV-LOX-1wt or CMV-LOX-1(1-261) plasmid (P<0.01 vs control). Fibroblasts transfected with CMV-LOX-1wt showed ox-LDL binding, whereas fibroblasts without transfection and those transfected with CMV-LOX-1(1-261) did not bind ox-LDL. Compared with untransfected cells, ox-LDL treatment (50 microg/mL, 24 hours) markedly induced the expression of the leukocyte adhesion molecules intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM)-1 as well as matrix metalloproteinase (MMP)-1 in cells transfected with CMV-LOX-1wt (P<0.05) but not in cells transfected with CMV-LOX-1(1-261). Concurrently, ox-LDL treatment enhanced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) (P<0.05 vs control) in CMV-LOX-1wt-transfected cells. These data suggest that in cardiac fibroblasts, ox-LDL binds to LOX-1 and activates p38 MAPK, followed by the expression of ICAM-1, VCAM-1, and MMP-1. Thus, fibroblasts transform into an endothelial phenotype on transfection with CMV-LOX-1wt and subsequent exposure to ox-LDL. This study provides a useful model system (plasmid-transfected fibroblasts) to study the molecular biology of LOX-1.

Animals↗
PubMed2004

Enhancement of hepatocyte growth factor-induced cell scattering in N-acetylglucosaminyltransferase III-transfected HepG2 cells.

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the synthesis of a bisecting GlcNAc residue of N-glycans, is thought to be involved in the function of glycoproteins such as growth factor receptors. We investigated the effects of the overexpression of GnT-III on the hepatocyte growth factor (HGF) receptor c-Met, a glycoprotein, in human hepatocarcinoma HepG2 cells. GnT-III activity was elevated about 250-fold in HepG2 cells stably transfected with the GnT-III gene, whereas no significant change in GnT-III activity was observed in mock transfectants. Cell scattering assay revealed that HGF-induced cell scattering was enhanced depending on the GnT-III activities in the GnT-III transfectants. Western blot analysis and E-PHA lectin blot analysis showed that the level of c-Met protein was the same in both transfectants; however, the bisecting GlcNAc residue on c-Met was detected only in the GnT-III transfectants. Although the peak level of c-Met phosphorylation was not different in both transfectants, the level of tyrosine phosphorylation of c-Met decreased more rapidly in the GnT-III transfectants than in the mock transfectants. Furthermore, HGF-induced extracellular-regulated kinase (ERK) phosphorylation was slightly higher in the GnT-III transfectants than in the mock transfectants. These results show that overexpression of GnT-III in HepG2 cells enhances HGF-induced cell scattering, which may result from, at least in part, enhancement of HGF-induced ERK phosphorylation.

Cell Line, Tumor↗
PubMed1992

Calcium requirement for protoplast transfection mediated by polyethylene glycol of Lactobacillus casei by PL-1 Phage DNA.

The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca2+. Sr2+ increased the transfection rate as well, but Ba2+, Mn2+, and Mg2+ did not. Co2+ and Zn2+ inhibited transfection. The simultaneous use of Ca2+ and Mg2+ increased the transfection efficiency. Impairment of transfection caused by lack of Ca2+ could not be reversed by the addition of Ca2+ later. A decrease in the Ca2+ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.

Bacteriophages↗
PubMed2006

Nucleofection is the most efficient nonviral transfection method for neuronal stem cells derived from ventral mesencephali with no changes in cell composition or dopaminergic fate.

Neuronal progenitor cells (NPCs) play an important role in potential regenerative therapeutic strategies for neurodegenerative diseases, such as Parkinson disease. However, survival of transplanted cells is, as yet, limited, and the identification of grafted cells in situ remains difficult. The use of NPCs could be more effective with regard to a better survival and maturation when transfected with one or more neurotrophic factors. Therefore, we investigated the possibility of transfecting mesencephalic neuronal progenitors with different constructs carrying neurotrophic factors or the expression reporters enhanced green fluorescence protein (EGFP) and red fluorescent protein (DsRed). Different techniques for transfection were compared, and the highest transfection rate of up to 47% was achieved by nucleofection. Mesencephalic neuronal progenitors survived the transfection procedure; 6 hours after transfection, viability was approximately 40%, and the transfected cells differentiated into, for example, tyrosine hydroxylase-positive neurons. Within the group of transfected cells, many progenitors and several neurons were found. To provide the progenitor cells with a neurotrophic factor, different isoforms of fibroblast growth factor-2 were introduced. To follow the behavior of the transfected cells in vitro, functional tests such as the cell viability assay (water-soluble tetrazolium salt assay [WST-1]) and the cell proliferation assay (5-bromo-2'-deoxyuridine-enzyme-linked immunosorbent assay) were performed. In addition, these transfected NPCs were viable after transplantation, expressed tyrosine hydroxylase in vivo, and could easily be detected within the host striatum because of their EGFP expression. This study shows that genetic modification of neural progenitors could provide attractive perspectives for new therapeutic concepts in neurodegenerative diseases.

Animals↗
PubMed2006

Parallel analysis of v-Src mutant protein function using reverse transfection cell arrays.

The conversion of the genomic information produced by the recent sequencing projects into a comprehensive understanding of the human proteome has yet to occur. A new technology that represents a potential bridge between genomics and proteomics is reverse transfection. Reverse transfection cell microarrays are produced by overlaying cDNA arrays with mammalian cells, generating localized clusters of transfected cells with each cluster overexpressing a unique protein. This miniaturized cell-based microarray format affords parallel functional analysis of thousands of cDNA constructs in a high throughput format. In this report we document the development of a co-transfection methodology for reverse transfection applications. The demonstrated high co-transfection efficiency with a "marker" plasmid encoding for GFP enables the identification of transfected cells and eliminates the need for epitope-tagged constructs in cell-based high throughput screening applications using reverse transfection. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein using automated fluorescence microscopy. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity. This reverse co-transfection approach is applicable for high throughput screening of both cDNA libraries and positional scanning recombinant protein libraries.

Cell Line↗
PubMed2000

Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity.

In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have shown that plasmid DNA can be entrapped in "stabilized plasmid lipid particles" (SPLP) using low levels (5-10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lipid at a 1:1 lipid ratio. The objective of this study was to construct SPLPs with increased cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5 mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 +/- 6 nm as determined by cryo-electron microscopy. The high plasmid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for in vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasing the cationic lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP showed maximum transfection activity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor cells was increased by approximately one magnitude as compared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at 4 degrees C no significant change in the transfection activity was observed over a one-year period. Thus this phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.

Animals↗
PubMed2005

Effect of deleted pancreatic cancer locus 4 gene transfection on biological behaviors of human colorectal carcinoma cells.

AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS: PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characterristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow- cytometry. RESULTS: PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population- doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P<0.01). The cloning efficiencies of DPC4+-SW620 cells (12%) were markedly lower than those of SW620 cells (69%) and PcDNA3.1-Sw620 cells (67%) (P<0.01). Compared with SW620 cells and PcDNA3.1-Sw620 cells, the G0-G1% of DPC4+-SW620 cells was obviously higher and the S% was markedly lower (P<0.05). Apoptosis rate of DPC4+-SW620 cells was significantly higher than that of SW620 cells and PcDNA3.1-SW620 cells. CONCLUSION: PcDNA3.1-DPC4 plasmid can be successfully re-constructed and stably transfected into human SW620 cells, thereby the cells can steadily express Smad4. DPC4 protein may regulate proliferation of colorectal carcinoma cells by inhibiting cell growth and inducing cell apoptosis.

Apoptosis↗
PubMed2005

Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells.

AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcription-polymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

Carcinoma, Hepatocellular↗
PubMed1999

Transfection of mammalian cells by the methods of receptor mediated gene transfer and particle bombardment.

The efficacy of currently developed methods for gene transfer into mammalian cells depends primarily on the transfection technique, and also on the type of targeted cells. Considering the importance of gene transfer in the creation of gene therapies, our study was aimed at the assessment of transfection capacity of receptor mediated gene transfer method (RMGT), and method of particle bombardment (helios gene gun system--HGG) in different normal and malignant mammalian cells ex vivo. In addition, the HGG was also assessed for its ability to transfect tumor cells of subcutaneous (s.c.) tumors in C57Bl/6 mice in vivo. Using RMGT an average ex vivo transfection rate of 35.7%, and 20.4% was achieved in malignant melanoma B-16, and human breast adenocarcinoma MCF7, respectively. However, in normal fibroblast L929 cells the transfection by RMGT succeeded only in 2.1% of the cells. On the other hand, the transfection efficacy of HGG was comparable in both malignant cell lines resulting in an average gene transfer to 9.6% of B-16 and 10.5% of MCF7 cells, while only 3.9% of normal fibroblasts were successfully transfected. Application of HGG for an in vivo gene transfer into s.c. B-16 melanoma tumors in C57Bl/6 mice resulted in a successful but limited transfection of the epithelium as well as of the superficially sited tumor cells. Taking into consideration both methods, RMGT is more appropriate for ex vivo transfection of cells, under the condition that target cells express a specific receptor for the molecule attached to the carrier. On the other hand, HGG is not complicated to use, no requirements for specific structures on target cells are necessary (potentially usable in different cells), and it has applicability in direct in vivo transfection processes.

Adenocarcinoma↗
PubMed2001

[Experimental study of in osteogenesis in duced by NIH-3T3 cells transfected with bone morphogenetic protein 3 gene].

OBJECTIVE: To investigate the gene expression, protein synthesis and inducing osteogenetic capacity of NIH-3T3 cells transfected with recombinant human bone morphogenetic protein 3 (rhBMP3) gene. METHODS: Gene recombination technique was used to clone the BMP3 cDNA into PcDNA3 vector directly, and to form a recombined BMP3-PcDNA3 cDNA, which was introduced into NIH-3T3 cells by means of lipofectamine transfer. The total RNA of transfected NIH-3T3 cells was then extracted to verify the mRNA expression of BMP3 using Northern blot method. BMP3 protein synthesis and AKP activity in the transfected cells was checked. Then, the NIH-3T3 cells transfected with BMP3 gene were injected into nude mouse muscle to evaluate the inducing osteogenesis in the muscle. RESULTS: BMP3 gene had been inserted into PcDNA3 vector successfully. There was BMP3 mRNA expression in the total RNA of NIH-3T3 cells with the peak level appearing in the 6th week after transfection, and BMP3 protein was found in NIH-3T3 cells, and the AKP level is much higher in the transfected NIH-3T3 cells than in non-transfected NIH-3T3 cells. Histologic examination revealed that there were a large number of chondrocytes and trabeculae seen in the nude mouse muscle 4 weeks after injection of the transfected NIH-3T3 cells. CONCLUSION: BMP3 is a protein that possesses osteogenetic effect. After transfection with BMP3-PCDNA3, BMP3 mRNA expression and protein formation can be found in NIH-3T3 cells, and the transfected NIH-3T3 cells have the effect of inducing osteogenesis in vivo.

3T3 Cells↗
PubMed2001

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2.

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic (alpha and alpha') and regulatory (beta) subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of 'free' CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemagglutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with alpha and beta subunits of Xenopus CK2, with the alpha' subunit of D. rerio, and with Xl CK2alphaA156, which although inactive can bind tightly to CK2beta, and with Xl CK2alphaE75E76, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10-20% of treated cells. Expression of CK2alpha or CK2alphaE75E76 in COS-7 cells caused an increase of 5-7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2beta, the activity increased further to 15-20-fold of the controls. Transfection of CK2beta alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2alphaA156 yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, co-transfection of CK2alpha or CK2alphaE75E76 with CK2alphaA156 caused a 60-70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2alpha subunits. These results can be interpreted as meaning that CK2alphaA156 is a dominant negative mutant that can compete with the other catalytic subunits for the CK2beta subunit. Addition of recombinant CK2beta to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2beta subunit is limiting in the extracts and that an excess of free CK2alpha has been produced in the transfected cells. Transfection of cells with CK2alphaE75E76 results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2alphaE75E76 to heparin increases considerably when it forms part of the holoenzyme CK2alpha2beta2.

Animals↗
PubMed2002

Protective effect against ischemia and light damage of iris pigment epithelial cells transfected with the BDNF gene.

PURPOSE: Brain-derived neurotrophic factor (BDNF) has been reported to protect retinal neurons against ischemia and light-induced damage. In the current study, the BDNF gene was transfected into iris pigment epithelial (IPE) cells of Long-Evans rats, and the neuroprotective ability of the IPE cells transfected with the BDNF gene against N-methyl-D-aspartate (NMDA)-induced neuroretinal cell death and against phototoxic damage was examined. METHODS: The level of BDNF mRNA and protein expressed in the transfected cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and by sandwich enzyme-linked immunosorbent assay (ELISA). The neuroprotective effects were determined by culturing BDNF gene-transfected IPE cells or nontransfected cells with neuroretinal cells in the presence of NMDA. The neuroprotective activity was also evaluated for the damage induced by constant exposure to light on the photoreceptors by transplanting BDNF gene-transfected IPE cells into the subretinal region of the superior half of the eye. RESULTS: BDNF gene-transfected IPE cells expressed higher levels of BDNF mRNA and protein than did nontransfected IPE cells. A significant increase in the protection against NMDA was observed in the neuroretinal cells cultured with BDNF-transfected IPE cells than in those cultured with nontransfected IPE cells (P = 0.0029) or with nontreated cells (P = 0.0010). The effect was partially attenuated by the addition of anti-BDNF antibody. Significant photoreceptor cell protection against injury from constant light was also observed by the subretinal transplantation of BDNF-transfected IPE cells when compared with those receiving transplants of nontransfected cells or vehicle injection. CONCLUSIONS: BDNF-transfected IPE cells demonstrated a neuronal rescue effect in both in vitro and in vivo experiments. IPE cells may be a potential source for autologous cell transplantation for some retinal diseases, and the transfection of the genes of neurotrophic factors into the transplanted cell may be a useful tool for delivering these factors to the retina.

Animals↗
PubMed2002

[Comparison of different methods of pEGFP-actin plasmid transfection to the endothelium].

OBJECTIVE: To screen the optimal transfection method for human umbilical vein endothelium, so as for us to establish a basic method for the further transfection with other objective genes. METHODS: The pEGFP-Actin plasmid was employed as an ectogenetic gene and was transfected into ECV-304 endothelium cells by lipofection, DEAE-dextran or electroporation transfection methods, respectively. The cell death rate and transfection rate after the transfection were compared among the three methods. RESULTS: (1) Twelve hours after the transfection, the cell death rate was 4% by all three methods, however, the cell death rate increased up to 50% at 60 hours after the transfection by DEAE-dextran. (2) The transfection rate of ECV-304 could reach 95% by all the three methods. But the fluorescent intensity was stronger in lipofection group compared with electroporation method. CONCLUSION: Gene transfection into ECV-304 with lipofection and electroporation methods exhibited better stability and repeatability.

Actins↗
PubMed2003

[Effect of HLCDG1 gene transfection on growth of lung carcinoma cells].

BACKGROUND & OBJECTIVE: HLCDG1, which locates in chromosome 5q33 (between D5S436 and D5S470),is a novel gene that our laboratory has cloned recently. The expression of HLCDG1 gene was significantly down-regulated or deleted in the primary lung carcinoma. This study was designed to observe if HLCDG1 has the potential to suppress growth of lung carcinoma cells. METHODS: The recombinant plasmid, pcDNA3.1(+)/HLCDG1, was constructed and subsequently transfected into A549 cells through liposome transfection. The A549 cells stably expressing HLCDG1 gene were established by G418 selection. RT-PCR was used to demonstrate the expression of HLCDG1 gene. Furthermore, the cell proliferation assay, the soft agar assay, and the tumorigenesis assay were used to analyze the malignant phenotype of the HLCDG1-transfected cells. RESULTS: The HLCDG1-transfected cells exhibited the expression of HLCDG1 mRNA by RT-PCR. The population double time (PDT) of HLCDG1-transfected group, vector-transfected group, and nontransfected group were 70.0 hours, 43.3 hours, and 39.5 hours, respectively; the difference between HLCDG1-transfected group and the other two groups was significant (P< 0.05). The colony formation rates of HLCDG1-transfected group, the vector-transfected group, and nontransfected group were 8.5%, 29.0%, and 35.0%, respectively. The rate of HLCDG1-transfected cells was markedly lower than those of the other two groups (P< 0.05). Moreover, these clones were injected into athymic nude mice. After 43 days, they were killed, and their tumors were isolated. These tumors weighed 0.120g, 0.612g, and 0.924g, respectively. CONCLUSION: The expression of HLCDG1 in A549 cells may have the potential to suppress tumor cell growth and the tumorigenesis of A549 cells transplanted in nude mice. These results suggested that HLCDG1 gene might be a good candidate of tumor suppressor gene correlated with lung carcinoma.

Animals↗
PubMed2003

[CIK cells acquired multidrug resistance and maintained cytotoxic activity to tumor cells after mdr1 gene transfection].

OBJECTIVE: To investigate whether the cytokine-induced killer (CIK) cells could acquire multidrug resistance and maintain the original cytotoxic activity after multidrug resistance (mdr1) genes transfection. METHODS: CIK cells were generated from peripheral blood cultured with IFN-gamma, CD(3) monoclonal antibody, IL-2, IL-1 and transfected with a plasmid (pHamdr) containing human mdr1 gene via electroporation. RT-PCR method was used to assay mRNA expression of mdr1 gene in transfected CIK cells, flow cytometry with anti-P-gp monoclonal antibody to detect P-glycoprotein (P-gp) expression on CIK cells membrane, and MTT assay to compare both the multidrug resistance to doxorubicin and colchicines and cytotoxic activity to human mammary cancer cell line MCF7 between transfected and non-transfected CIK cells. RESULTS: mdr1 expression was detected in the transfected CIK cells. There was a strong expression of P-gp on the transfected CIK cells and the percentages of P-gp positive cells were 21% - 37% (average 27%). The IC(50) of transfected CIK cells to doxorubicin was 22.3 - 45.8 times and 6.7 - 11.35 times to colchicines of those of non-transfected CIK cells. The cytotoxic activity to MCF7 remained unchanged (P > 0.05). CONCLUSION: It demonstrated that CIK cells transfected with mdr1 gene via electroporation could express multidrug resistance successfully without changes of cytotoxic activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗