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At least 73 records · Page 4Linked to original sources
PubMed1992

Study of mechanisms of electric field-induced DNA transfection. IV. Effects of DNA topology on cell uptake and transfection efficiency.

Electric parameters and solvent conditions are known to influence the efficiency of DNA transfection of cells by a pulsed electric field (PEF). A previous study (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO (Eur. Mol. Biol. Organ.) J. 1:841-845) has indicated that DNA topology is also an important determinant. We report an investigation of the PEF induced uptake, stability, and expression of three different topological isomers, circular supercoiled (scDNA), circular relaxed (crDNA), and linearized (lnDNA) forms of the plasmid pBR322, by Escherichia coli strain JM105. Monomeric pBR322 prepared by the electroelution from an agarose gel was in the supercoiled form. Treatment of the scDNA with wheat germ topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of scDNA by a restriction endonuclease, EcoRI or Hind III, linearized the DNA. The MgCl2-dependent bindings of all three forms of DNA to the cell surface were indistinguishable. So was the PEF induced cell uptake. In contrast, the transfection efficiency (TE) for the scDNA and the crDNA were high (approximately 2 x 10(8) micrograms-1 DNA at neutral pH), whereas that for the lnDNA was approximately five orders of magnitude lower (less than 1 x 10(3) micrograms-1 DNA). Analysis by agarose gel electrophoresis indicated that the PEF loaded ln DNA was degraded by the host cell within 3 h. However, the loaded scDNA and the crDNA were stable and expressed in the cytoplasm. We conclude that first, the PEF induced DNA entry into E. coli did not depend on the topology of the DNA. As cellular uptake of DNA also correlated with the surface binding, these data support electrophoresis of surface bound DNA as the dominating mechanism for the DNA entry. Second, the variations of TE for different topological forms of DNA reflected their relative stability in the host cells. Third, since the loaded DNA could be either rapidly degraded by the host enzyme or expressed, they were unlikely coated with a layer of protective lipid membrane. Thus, PEF induced cellular uptake of DNA is unlikely by the endocytotic mechanisms as was reported previously for the liposomes (Chernomordik, L. V., A. V. Sokolov, and V. G. Budker. 1990.Biochim. Biophys. Acta. 1024:179-183).

Ampicillin Resistance↗
PubMed1994

Malignant transformation of a human fibroblast cell strain by transfection of a v-fes oncogene but not by transfection of a gag-human c-fes construct.

To determine whether the human c-fes gene, a homologue of the feline v-fes oncogene, can play a role in the malignant transformation of human fibroblasts, we transfected a near-diploid, infinite life span, growth factor-independent, human fibroblast cell strain, MSU-1.2, with plasmids carrying a fes gene along with a selectable marker. The fes gene was either the v-fes oncogene from the Gardner-Arnstein strain of feline sarcoma virus or a chimeric construct in which 835 base pairs representing exons 10-19 from the human c-fes proto-oncogene had been substituted for the corresponding feline sequence in the v-fes oncogene. The transfected cells were selected in appropriate medium, and a number of drug-resistant clones were isolated, and the progeny cells were assayed for fes expression by immunoprecipitation analysis. Six independent clones that expressed the v-fes protein and four that expressed the gag-c-fes protein were further characterized. The former exhibited anchorage independence and formed progressively growing, invasive, spindle cell sarcomas in athymic mice after only a short latency period. The latter strains were not anchorage independent and did not form tumors in athymic mice. These results show that the v-fes oncogene can malignantly transform an infinite life span human fibroblast cell strain, but the human c-fes gene cannot.

Animals↗
PubMed1996

E-cadherin transfection down-regulates the epidermal growth factor receptor and reverses the invasive phenotype of human papilloma virus-transfected keratinocytes.

The human papillomavirus type 16 (HPV-16), the type most often associated with cervical cancer, immortalizes primary keratinocytes and inhibits serum/calcium-stimulated differentiation in culture. In this study, we have used a model of keratinocyte immortalization based upon HPV-16 to analyze perturbation of function and expression of E-cadherin, a Ca(2+)-dependent cell-cell adhesion molecule expressed by normal keratinocytes, and its associated proteins. An immortalized keratinocyte cell line generated by cotransfection with HPV-16 E6 and E7 showed decreased membrane E-cadherin expression and redistribution of alpha-, beta-, and gamma-catenin from the undercoat membrane to the cytoplasm. No changes in the level of expression were seen. Selection of the immortalized keratinocyte cell line for resistance to differentiation generated a more transformed cell line with an invasive phenotype, down-regulated E-cadherin and alpha-catenin, and up-regulated the epidermal growth factor receptor (EGFr). Transfection of an E-cadherin expression construct into the differentiation-resistant cell line restored membrane-bound E-cadherin and catenin expression, down-regulated the EGFr, and reversed the invasive phenotype. These results indicate that overexpression of the EGFr correlates with perturbation of the E-cadherin/catenin complex seen in the HPV-16 E6- and E7-transfected keratinocytes and may underlie a functional interaction between growth-regulatory factors and adhesion molecules (E-cadherin/catenin).

Animals↗
PubMed1974

Protease-sensitive transfection of Bacillus subtilis with bacteriophage GA-1 DNA: a probable case of heterologous transfection.

The host bacterium of bacteriophage GA-1, Bacillus sp. G1R, was compared with respect to its taxonomic relationship to Bacillus subtilis, B. licheniformis, and B. pumilis. The physiological-biochemical properties of Bacillus sp. G1R are equal to those of B. licheniformis, but the thermal denaturation midpoint of G1R DNA differs by 3 C and the buoyant density by 0.005 g/cm(3) from that of B. licheniformis. Transformation with G1R donor DNA was neither observed in B. licheniformis nor in B. subtilis-competent recipients. Bacteriophage GA-1 shows neither infectivity on B. licheniformis nor on B. subtilis. However, infection of competent B. subtilis cultures with phenol-extracted GA-1 DNA results in the production of infective GA-1 particles. The transfecting activity of GA-1 DNA is destroyed by treatment with proteolytic enzymes. Resistance of transfecting DNA to inactivation by trypsin develops earlier than that to inactivation by DNase. Protease-treated GA-1 DNA competes with transforming DNA to approximately the same extent as does untreated GA-1 DNA, suggesting that uptake of GA-1 DNA is not affected by protease treatment. CsCl density gradient centrifugation reveals that the density of trypsinized GA-1 DNA is 0.004 g/cm(3) greater than that of untreated DNA.

Bacillus↗
PubMed2003

Malaria transfection and transfection vectors.

Malaria remains a leading cause of death due to infectious disease. The completion of the Plasmodium falciparum genome sequencing project and release of preliminary proteomics data have significantly increased our understanding of the biology of this organism. Nonetheless, additional tools for functional analysis of this massive amount of information are now indispensable to further understand the basic biology of this parasite. The genetic manipulation of specific genes via the use of plasmid constructs and transfection represents one such tool.

Animals↗
PubMed1974

Transfection of Escherichia coli spheroplasts. IV. Transfection of rec+ and rec minus spheroplasts by native, denatured, and renatured T5 bacteriophage DNA after repair of single-strand breaks by polynucleotide ligase.

Transfection of Escherichia coli spheroplasts by native T5 phage DNA was not affected by treatment with polynucleotide ligase. Denatured T5 phage DNA infectivity, only 0.1% of the native DNA level, was increased slightly by polynucleotide ligase treatment. Renatured T5 phage DNA infectivity was also increased slightly by polynucleotide ligase treatment. To form an infective center with rec(+) spheroplasts, 1.6 to 2.1 native T5 phage DNA molecules were required; however, 1.4 T5 phage DNA molecules were required to form an infective center with recA(-)B(-) spheroplasts, and one molecule was sometimes sufficient for rec B(-) spheroplasts. Polynucleotide ligase treatment of T5 phage DNA had no effect on these parameters. Thus, the single-strand interruptions of T5 phage DNA are probably not essential to the survival of the parental T5 phage DNA, and T5 phage DNA, especially the denatured form, is highly sensitive to some nucleases in E. coli spheroplasts.

Coliphages↗
PubMed1991

Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

HLA-B27-transfected (Salmonella permissive) and HLA-A2-transfected (Salmonella nonpermissive) human monocytic U937 cells differ in their production of cytokines.

The cytokine secretion of the Salmonella-permissive, HLA-B27-positive U937 cells was examined, as it was previously shown that these cells kill Salmonella less efficiently than controls. Salmonella-permissive U937 cells showed upregulated production of interleukin 10 and to a lesser extent tumor necrosis factor alpha. HLA-B27-associated modulation of cytokine responses may have importance in the pathogenesis of reactive arthritis.

Arthritis, Reactive↗