Efficient transient and stable expression in mammalian cells of transfected genes using erythrocyte ghost fusion

We have modified the erythrocyte ghost fusion transfection method (F.C. Wiberg, P. Sunnerhagen, K. Kaltoft, J. Zeuthen, and G. Bjursell (1983) Nucleic Acids Res. 11, 7287) by changing the resealing buffer to a Ca2+-containing ghost shrinkage solution, which considerably enhances DNA loading and transfection efficiency. In addition, we have demonstrated the generality of the transfection method by successfully transfecting different cell types, including lymphoid cells, obtaining both transient and long-term expression.     

Hypergravity signal transduction and gene expression in cultured mammalian cells

A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.     

20-Hydroxyecdysone increases levels of transient gene expression in transfected Drosophila cells.

Methods for increasing the transient level of expression of transfected DNA in cultured Drosophila cells have been examined. Here we show that cells exposed to the steroid hormone 20-hydroxyecdysone for 48h prior to transfection show an 4 to 5 fold increase in the levels of expression of transfected DNA. By analysis, in situ, this increase appears to be due to an increase in the number of cells expressing the transfected DNA. The stimulation in levels of expression is not correlated to any specific DNA sequence, nor does it occur if cells are exposed to hormone post-transfection.     

Neutral selection of transfected mammalian cells using tissue plasminogen activator gene expression

The gene that produces the proteolytic enzyme, tissue plasminogen activator (tPA), was used as a selectable marker for transfection. Cells that were successfully transfected with plasmids containing a gene for tPA (tpa) produce plaques in the caseinolysis assay. Two tpa-containing plasmids were constructed and used to transfect a variety of cell types. One plasmid contained the promoter region of simian virus 40 and the other contained the Moloney murine leukemia virus promoter. No significant difference in transfection frequencies was found for the two plasmids. However, 80% of the cell types tested produced plaques. This system allows for the identification and isolation of transfected cells under conditions that are not lethal to nontransfected cells.     

MDR1 gene expression enhances long-term engraftibility of cultured bone marrow cells

Primitive hematopoietic stem cells are responsible for long-term engraftment in irradiated host. Here, we report that multi-drug resistance 1 (mdr1) gene expressing primitive hematopoietic cells were multiplied in ex vivo culture, with the support of extracellular matrix components and cytokines. About 20-fold expansion of total nucleated cells was achieved in a 10-day culture. Lin(-)Sca-1(+) and long-term culture-initiating cells were increased by 54- and 26-fold, respectively. Expanded cells were long-term multi-lineage engraftible in sub-lethally irradiated mice. Donor-derived peripheral blood chimerism was significantly higher (73.2+/-9.1%, p<0.01) in expanded cells than in normal and 5-flurouracil-treated bone marrow cells. Most interestingly, the expression of mdr1 gene was significantly enhanced in cultured cells than in other two sources of donor cells. The mdr1 gene was functional since expanded cells effluxed Hoechst 33342 and Rh123 dyes. These results suggest that primitive engraftible stem cells can be expanded in the presence of suitable microenvironments.     

5-Azacytidine facilitates osteogenic gene expression and differentiation of mesenchymal stem cells by alteration in DNA methylation

Mesenchymal stem cells (MSCs) are considered to be one of the most promising therapeutic cell sources as they encompass a plasticity of multiple cell lineages. The challenge in using these cells lies in developing well-defined protocols for directing cellular differentiation to generate a desired lineage. In this study, we investigated the effect of 5-azacytidine, a DNA demethylating agent, on osteogenic differentiation of MSCs. The cells were exposed to 5-azacytidine in culture medium for 24 h prior to osteogenic induction. Osteogenic differentiation was determined by several the appearance of a number of osteogenesis characteristics, including gene expression, ALP activity, and calcium mineralization. Pretreatment of MSCs with 5-azacytidine significantly facilitated osteogenic differentiation and was accompanied by hypomethylation of genomic DNA and increased osteogenic gene expression. Taking dlx5 as a representative, methylation alterations of the “CpG island shore” in the promoter caused by 5-azacytidine appeared to contribute to osteogenic differentiation.     

Growth rate suppression of cultured mammalian cells enhances protein productivity

Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitively growth-suppressed accumulated the mRNA of IgG₁ which is reported stable, and IgG₁ production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM ₁ chain and cultured at nonpermissive temperature to enhance production of ₁. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.Abbreviations IL6 recombinant human interleukin-6 - TGF- recombinant human TGF-₁ - X63.653-P3X63 Ag8.653 myeloma     

Camptothecin enhances random integration of transfected DNA into the genome of mammalian cells

In order to study the involvement of DNA topoisomerase I (top1) in recombination, we examined the effect of the anti-neoplastic drug camptothecin, which selectively poisons top1 by trapping top1-cleavable complexes on integration of exogenic vector into the genome of mammalian cells. We transfected mouse F9 teratocarcinoma cells as well as Chinese hamster V79 cells with a plasmid carrying a selectable neo gene treated with camptothecin, and determined the frequency of neo+ (G418(R)) colonies. We found that treatment with camptothecin for as short a time as 4 h after electroporation resulted in a 4- to 33-fold stimulation of plasmid integration into the recipient genome via non-homologous recombination. These results imply that top1-cleavable complexes trapped by camptothecin could be potentially recombinogenic structures and could stimulate non-homologous recombination in vivo, promoting the integration of transfected plasmids into mammalian genome.     

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.     

Aberrant gene expression in cultured mammalian bone cells demonstrates an osteoblast defect in osteopetrosis

Osteopetrosis is a skeletal condition in which a generalized radioopacity of bone is caused by reduced resorption of bone by osteoclasts. However, it has recently been shown that during skeletal development in several osteopetrotic rat mutations specific aberrations occur in gene expression reflecting the activity of the bone forming cells, osteoblasts, and the development of tissue organization. To evaluate their pathogenetic significance, progressive osteoblast differentiation was studied in vitro. Primary cultures of normal osteoblasts undergo a sequential expression a cell growth and tissue-related genes associated with development of skeletal tissue. We report that osteoblast cultures can be established from one of these mutants, toothless; that these cells in vitro exhibit similar aberrations in gene expression during cell proliferation and extracellular matrix formation and mineralization observed in vivo; and that an accelerated maturation sequence by mutant osteoblasts mimics the characteristic skeletal sclerosis of this disease. These data are the first direct evidence for an intrinsic osteoblast defect in osteopetrosis and establish an in vitro model for the study of heritable skeletal disorders. © 1994 Wiley-Liss, Inc.     

四种启动子调控RFP报告基因在家蚕细胞(Bm-e-HNU5)内的瞬时表达

以红色荧光蛋白基因（RFP）为报告基因,构建含4种不同启动子的重组表达质粒,用脂质体介导法转染家蚕Bombyx mori细胞（Bm-e-HNU5）,观察家蚕细胞质肌动蛋白4基因启动子（A4）、α微管蛋白基因启动子（α-tub）、蚕丝心蛋白重链基因启动子（Fib）和家蚕核型多角体病毒早期即刻蛋白基因启动子（IE）4种启动子调控RFP报告基因在家蚕细胞内的瞬时表达情况。构建的重组表达质粒pDsRed-α-tub、pDsRed-A4、pDsRed-IE和pDsRed-Fib经双酶切和PCR鉴定正确无误。转染和转录实验结果表明,除了pDsRed-A4外,其他3种重组质粒在Bm-e-HNU5细胞中都得到高转染率,α-tub、IE和Fib可依次增强RFP报告基因在家蚕细胞内的瞬时表达活性。     

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.     

SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture

A transient transfection process was established using a novel 'in-house' developed transfection reagent, Ro-1539. It allows rapid production of large quantities of various recombinant proteins. Here we describe the transient expression of the secreted human placental alkaline phosphatase (SEAP) by HEK293EBNA and CHO cells in serum-free suspension culture. Unexpectedly, high expression levels of SEAP (150 μg/ml) were found 3–4 days post-transfection when placental alkaline phosphatase (AP) was used as the reference enzyme. To confirm these data, an SDS–PAGE analysis was performed and the visible SEAP protein band (MW of 65 kDa) was compared with co-migrated purified placental AP protein as reference. The scanning analysis of the gel showed that SEAP, a truncated form of AP, has a higher specific activity than the purified placental AP. A correction factor was introduced permitting a direct comparison of placental AP activity with the expression levels of SEAP. Scale-up of the transfection system from spinner flask to bioreactor was simple and straightforward, resulting in similar yields of SEAP. Finally, the effectiveness of Ro-1539 was compared to that of other transfection reagents. This revised version was published online in July 2006 with corrections to the Cover Date.     

Automation of large scale transient protein expression in mammalian cells

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI? cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.     

Retrovirus-mediated gene expression in mammalian cells.

Significant advances have been made in precisely defining the elements in the Moloney murine leukemia virus genome responsible for tissue-restricted expression. This knowledge should lead to improved expression vectors for gene transfer in mammalian cells. In the past year, retrovirus-mediated gene expression in a diverse range of cell types has been reported. These cells have been used to study gene transfer relevant to a range of inherited diseases.     

Phage particle-mediated gene transfer to cultured mammalian cells

Recombinant phage particles carrying the thymidine kinase (TK) gene of herpes simplex virus type 1, coprecipitated with calcium phosphate, efficiently transformed mouse Ltk- cells to the TK+ phenotype. The conditions necessary to achieve high efficiency of transfer of the TK gene by phage particle-mediated gene transfer were investigated. Of the parameters examined, the pH of the buffer used for coprecipitation of phage particles with calcium phosphate, the length of time of coprecipitation, and the length of the adsorption period were found to alter the transfer efficiency significantly. The optimal pH was 6.87 at 25 degrees C. The other optimal values for these parameters were as follows: coprecipitation time, 7 to 20 min; adsorption time, 18 to 30 h. Treatment with dimethyl sulfoxide, glycerol, or sucrose did not enhance gene transfer. The optimal conditions yielded about 1 transformant per 10(5) phage particles per 10(6) cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 x 10(7) phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 10(10) phage particles per dish, did not significantly affect the number of transformants.