Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells.

The nucleoprotein structures formed on various plasmid expression vectors transfected into mammalian cells by both the calcium phosphate and DEAE-dextran methods have been studied. We demonstrate by a variety of means that mammalian cells are capable of rapidly assembling non-integrated circular plasmids (both replicating and non-replicating) into typical "minichromosomes" containing nucleosomes with a 190 bp repetitive spacing. Treatment of recipient cells with sodium butyrate for a short period of time (12-16 h) immediately following transfection markedly increased the DNase I digestion sensitivity of the newly assembled plasmid chromatin. Furthermore, minichromosomes isolated from such butyrate-treated cells are depleted in histone H1 and contain highly acetylated forms of histone H4. These findings are entirely consistent with our earlier speculation (Gorman et al., Nucleic Acids Res. 11, 1044; 1983) that appropriate butyrate treatment might stimulate transient expression of newly transfected genes by facilitating their assembly into an "active" type of chromatin structure.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation

The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production. This revised version was published online in August 2006 with corrections to the Cover Date.     

鲤鱼金属硫蛋白基因启动区功能的研究

以氯霉素乙酰化酶作为报讯基因、利用草鱼肾培养细胞瞬时表达系统,对已克隆的鲤鱼金属硫蛋白基因５’－调节区１．６ｋｂ的序列进行了功能分析。从顺式效应和反式效应研究证明：所克隆的鲤鱼ＭＴ基因５‘－调节区具有典型ＭＴ启动子的特性实验发现哺乳动物病毒ＳＶ４０增强子要以加强鱼类ＭＴ启动子的活性,提示在系统进化上鱼类基因不但存在增强子元件,并具有哺乳动物增强子相似的作用方式,而且作用于增强子的反式效应因子也存在     

Sodium butyrate activates human immunodeficiency virus long terminal repeat--directed expression

To study the effect of sodium butyrate on human immunodeficiency virus (HIV) long terminal repeat (LTR)--directed expression, we constructed a chimeric plasmid (pLTR-CAT) in which the LTR sequences derived from a molecular clone of HIV were fused to the chloramphenicol acetyltransferase (CAT) gene. We used transient expression assays in transfected tissue culture cells to monitor the activity of the LTR. The expression of the pLTR-CAT plasmid was activated when the cells were exposed to butyrate after transfection. The magnitude of butyrate-induced increase was linear up to an 8 mM concentration and was different with regard to the target promoters used. Recombinant plasmids linked to marker genes may be useful models for studying the effects on HIV of various agents of chemical and biological origin.     

Long duration electroporation for achieving high level expression of glucocorticoid receptors in mammalian cell lines

A method is presented that utilizes long duration electroporation (LDE) to more efficiently introduce DNA into mammalian cell lines than standard electroporation techniques. With SV40-based vectors, more than 550,000 glucocorticoid receptors (GRs) per cell could be obtained in COS-7 cells with good cell survival. In experiments with a CMV-driven vector expressing an enhanced Green Fluorescent Protein (EGFP), 54% of the cells were transfected, and 77% of EGFP positive cells expressed EGFP at moderate to high levels. In cell lines not containing the large T antigen, a CMV-driven vector for the GR was superior to the SV40-based vector. In EDR3, DG44, and CV-1 cell lines approximately 220,000, 190,000 and 150,000 GRs/cell were obtained, respectively. Transfection efficiency of the EGFP vector ranged from 44 to 55% for the three cell lines. Cortisol treatment of COS-7 and DG44 cultures cotransfected with vectors expressing the GR and a GRE driven luciferase gene produced 4 to 12 times more enzyme activity per plate with LDE than conventional electroporation protocols. LDE allows transient overexpression of proteins in COS-7 cells at the high levels generally achieved by mammalian overexpression systems only in stable cell lines.