电脉冲介导基因在小鼠杂交瘤细胞的高效转移

本文应用电脉冲介导基因转移法,获得pSV2-neo基因在小鼠杂交瘤细胞(B889)中的高效转移和稳定表达。研究了电场强度、脉冲宽度、脉冲次数等电场参数对基因转化频率的影响和小鼠杂交瘤细胞最适电转移条件。     

Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization

Dispase-treated murine hybridoma cells (SP2/0-Ag14) were transfected with the G418 resistance gene bearing plasmid pSV2-neo by electropermeabilization with a high degree of efficiency. The cells were subjected to intermittent multiple high-voltage short duration (5 microsecond) DC pulses at intervals of 1 min in a weakly conducting medium followed by selection in G418-containing medium. The transfection medium, temperature, pulse duration, and voltage were empirically determined by preliminary electropermeabilization experiments. Increasing the number of pulses resulted in a higher percentage of transfected cells, but a decrease in the number of viable cells, with the optimal transfectant yield resulting when five pulses of 10 kV/cm were administered. This method allows the rapid and efficient injection of DNA into mammalian cells, and permits the rapid production of stable, drug resistant hybridoma cell lines for use in subsequent fusion experiments.     

Enhancement of hybridoma production by medium supplemented with murine ascitic fluid

We have greatly improved the yield after cell fusion of antigen-specific monoclonal antibody-secreting murine hybridomas by substitution of Sp2/0 ascites for fetal bovine serum (FBS) in the culture medium. As a medium supplement for established cultures, Sp2/0 ascites at 2.5% in Dulbecco's modified Eagle's medium (DMEM) provided growth characteristics similar to media containing 10% or 20% FBS. All culture parameters associated with hybridoma fusion experiments, however, were advantageously affected in ascites-supplemented cultures. Clonal growth of hybridomas from the single cell stage was enhanced at least two-fold over 20% FBS-supplemented medium. Following fusion, both the number of colonies and hybridoma growth rates were substantially increased for ascites-containing cultures. Most importantly, the number of antigen-specific antibody-secreting hybridomas was increased in Sp2/0 ascites supplemented cultures, five-fold in the eight fusion experiments presented here. This improved performance compared to FBS-supplemented medium is reproducible from lot to lot of ascites.     

DNA topoisomerase II inhibitors enhance random integration of transfected vectors into human chromosomes

To study the involvement of DNA topoisomerase (topo) II on nonhomologous (illegitimate) recombination, we examined the effect of topo II inhibitors on random integration of exogenous vectors into human chromosomes. We transfected human cell lines PA1, HeLa and EJ-1 with linearized plasmid pSV2neo by electroporation, treated with topo II inhibitors and determined the frequency of Geneticin-resistant (G418^r) colonies. We found that three topo II inhibitors, etoposide (VP-16), ICRF-193 and amsacrine (m-AMSA), greatly enhanced the frequency of G418^r colonies. These effects were maximally expressed by as little as 12 hrs treatment with the drugs. Similar enhancements were found with different vectors (closed-circular and linear), different cell types, or by different transfection methods (calcium precipitation and lipofection). In contrast, the inhibitor treatments did not affect the transient expression of chloramphenicol acetyltransferase and β-galactosidase activity following transfection with pSV2CAT and pCH110, respectively. Southern blot analysis revealed that the integration pattern of transfected pSV2neo into PA1 chromosomes was random and not characteristic for each inhibitor: These results suggest that topo II inhibitors directly act at a nonhomologous recombination reaction, promoting the integration process of transfected vectors into human chromosomes. We discuss the enhancement mechanism with a special emphasis on DNA strand breaks induced by the inhibitors.     

Establishment of transformed swine fibroblast cell lines using SV40 large T antigen

Summary Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.     

Effects of culture media on murine hybridomas: definition of optimal conditions for hybridoma viability, cellular proliferation, and antibody production

Different cell culture media were compared for their ability to support and promote the growth of stable hybridoma cell lines derived from three commonly used parental murine myelomas. Supplemented Dulbecco's modified Eagle's media (DMEM) and RPMI 1640 media were studied. The DMEM-based media were found to support greater numbers of cells for longer time periods than were the RPMI 1640-based media. Aminopterin supplemented medium was shown to be significantly less effective in supporting hybridoma reproduction and viability than medium without aminopterin. Antibody levels were directly related to cell concentration and viability regardless of the medium used for the hybridoma culture. An optimally formulated DMEM-based medium is suggested as the medium of choice for hybridoma propagation and maintenance.     

High-efficiency stable gene transfection using chloroquine-treated Chinese hamster ovary cells

We describe a highly efficient stable gene transfection procedure for Chinese hamster ovary (CHO) cells using a modification of the calcium phosphate-DNA coprecipitation method. We have found that treatment of CHO cells with chloroquine increases the efficiency of gene transfer by up to 20-fold (from approx. 0.01% to approx. 0.2%) when transfection is done using the pSV2-neo plasmid. The optimized transfection procedure requires that CHO cells to be incubated with calcium phosphate-DNA coprecipitate and chloroquine (100 µM) for a total of 16 h. By using high-molecular-weight human genomic DNA as a DNA source for transfection, we show that this procedure is equally efficient for stably transferring a much larger gene, such as the 49-kb human hypoxanthine phosphoribosyltransferase gene.     

In vitro simian virus 40 large tumor antigen expression correlates with differential immune responses following DNA immunization

Simian virus 40 (SV40) contains an essential protein, large tumor antigen (Tag), which assists in viral replication and causes cell transformation and immortalization. Our laboratory has examined plasmid DNA, expressing SV40 Tag under two different promoters, for use in potential cancer vaccination strategies. One plasmid, pSV3-neo, failed to induce SV40 Tag antibody, produced a weak cell-mediated response, and only partial protection in murine experimental tumor challenge systems. The second plasmid, pCMV-Tag, induced antibodies to SV40 Tag, produced a robust cell-mediated response, and invoked complete tumor immunity in vivo. The induction of CD4+ and CD8+ T cell responses following plasmid DNA immunization and tumor cell challenge reflected a type 1 cytokine secretion profile. Our hypothesis for this differential immune response is that pCMV-Tag exhibits a higher level of transgene expression due to a more efficient promoter. We determined that pCMV-Tag levels of SV40 Tag mRNA and protein expression were higher when compared to pSV3-neo. A threshold amount of SV40 Tag may be required to stimulate antibody production and provide complete systemic tumor immunity.     

Replication of pSV2-gpt in COS-1 cells: Stability of plasmid DNA in the presence and absence of biochemical selection

We have previously demonstrated that COS-1 cell lines transformed by pSV2-gpt and maintained under biochemical selection replicate multiple copies of extrachromosomal plasmid DNA (1). We have now examined the replication and stability of this DNA in a representative cell line. In situ hybridization analyses revealed that intense replication of pSV2-gpt occurs in only a small subpopulation of cells and results from bursts of plasmid replication that occur periodically and spontaneously in the cell population. This suggests that COS-1 cells are only semipermissive for pSV2-gpt replication. No correlation was observed between levels of pSV2-gpt replication and the presence or absence of biochemical selection for the Gptmarker. However, growth of cells under nonselective conditions led to a rapid and progressive loss of pSV2-gpt DNA. This loss correlated with segregation of Gpt ^– revertants that lacked detectable plasmid sequences. Hence, maintenance of pSV2-gpt in the cell line was dependent on continuous biochemical selection. Stable replication of pSV2-gpt could be observed as late as four months after transfection, suggesting that this system might be useful for propagation of cloned DNA in COS-1 cells for extended periods of time. However, by nine months, extensive rearrangements of pSV2-gpt sequences were detected, indicating ultimate instability of the plasmid in the host cells.     

Factors influencing efficiency and reproducibility of polybrene-assisted gene transfer

A systematic investigation of factors influencing the efficiency of polybrene-assisted gene transfer for both transient and stable foreign gene expression was carried out utilizing NIH 3T3 fibroblasts as prototypic recipients for the plasmid expression vectors pSV2cat and pSV2neo. While transfection cocktail composition and cell density, in addition to polybrene exposure conditions and exogenous DNA concentration, each played an important role, the key determinant to achieving excellent transfection efficiency proved to be the DMSO treatment regimen. Under optimal conditions, the yield of colonies resistant to the neomycin analog, G418, increased linearly at the rate of 10 clones/ng of input (native form I pSV2neo) DNA up to a plasmid concentration of 50 ng, whereupon the dose-response for colony recovery became semilogarithmic. The incidence of stable transformants was doubled by linearization of the vector DNA, whereas the addition of carrier DNA to the transfection cocktail was without effect until present at concentrations above 10-fold molar excess, at which point the efficacy of gene transfer declined rapidly. Combined Southern and dot-blot analyses of transformed cell DNA demonstrated that the polybrene-DMSO procedure led to the stable integration of relatively few copies of the marker gene in each transformant; the actual number varied from 1–3 to 10–15 per host genome, depending on the concentration of pSV2neo DNA added. The potential for the adaptation of this DNA transfection procedure for general use with other mammalian cell types, as well as its technical strengths and weaknesses, is discussed.     

电穿孔法高效转染COS-7成纤维细胞系

目的　以电穿孔法转染COS 7细胞系以得到较高的转染率。方法　以CMV/LacZ质粒转化COS 7细胞系 ,48小时后用X gal染色。观察不同的穿孔电压 (6 0 0V ,5 5 0V ,5 0 0V ,45 0V ,40 0V)、时间常数 (40us、10 0us)、操作温度 (室温 ,4℃ )、质粒浓度 (5us/ml,2 5us/ml)和细胞悬液体积 (2 0 0ul、40 0ul)对转染率的影响。结果　利用Eppendorf公司的Multiporator及低渗透压缓冲液转化COS 7细胞的最优条件是 :转化体积 40 0 μl,细胞密度 2× 10 6cells/ml,场强 2 0 0 0V/cm ,时间常数 10 0us,室温质粒浓度2 5 μg/ml。条件优化后COS 7转染率可达 85 %。 结论　优化的电穿孔法可得到较高的COS 7转染率。     

Somatic cell hybrid selection with a transfectable dominant marker

A recombinant plasmid vector, pSV2-neo, coding for resistance to neomycin and the related antibiotic G-418, was transfected into the mouse myeloma line X63-Ag8.653 by a modification of the protoplast fusion technique. The time interval required to obtain 10⁶ G-418 resistant cells was 20 days and the efficiency was 10^–4–10^–5, which represents a significant advantage over classical methods of selecting mutant cells bearing a dominant selection marker. To investigate the efficiency of this marker in somatic cell hybrid selection, these cells were fused to the human myeloma line U-266 and the hybrids were selected either in HAT + G-418 or HAT + ouabain. The pSV2-neo vector was as efficient as ouabain as a dominant marker with respect to the number of viable hybrid colonies selected and their levels of immunoglobulin secretion. The reciprocal experiment was also performed: HAT-sensitive, mutant U-266 cells were transfected with pSV2-neo, clones selected in G-418 and fused with X63-Ag8.653 cells, and hybrids selected in ouabain plus G-418, yielding HAT-sensitive hybrid heteromyelomas that were effective fusion partners with human B lymphocytes.     

Transfection optimization for primary human CD8+ cells

Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by > 95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.     

The in vivo clearance of Ha-ras transformants by natural killer cells

The experiments in this study were designed to test the hypothesis that natural killer (NK) cells play a role in host surveillance against early neoplastic changes in the malignant process. C3H 10T1/2 mouse fibroblasts were transfected with a pSV2-neo plasmid vector which contains EJ, the mutated c-Ha-ras, regulated by its own promoter. Control cells were transfected with pSV2-neo alone and did not contain the ras gene. Oncogene-transfected cells were compared with control cells for lung colony formation following tail vein injection into C3H mice. Intravenous injection of ras-transfected 10T1/2 cells induced marked lung colony formation in vivo, whereas C3H 10T1/2 parental lines or 10T1/2 cells transfected with pSV2-neo alone induced no lung colonies in C3H mice. The colonising potential of ras transfectants could be decreased by augmentation of NK activity by injection of polyinosinic cytidylic acid and increased by depletion of NK effectors with anti-asialo GM₁. Experiments with beige mice demonstrated that the mortality of syngeneic, NK-deficient C3H-bg/bg mice injected with ras tranfectants was significantly greater than similarly treated NK-normal C3H-+/bg littermate controls. The results support the view that NK cells are capable in vivo of recognizing early defined stages in the neoplastic process initiated by oncogenes.     

Regulation of G418 selection efficiency by cell-cell interaction in transfection

We attempted to establish the optimum conditions for the calcium phosphate (CaPO₄) precipitation protocol by counting G418 resistant (G418^r) colonies after transfection of pSV2-neo DNA into BALB 3T3 cells. The amount and molecular size of carrier DNA, number of plating cells, treatment period of DNA-CaPO₄ precipitates and expression time of G418 selection were found to be important factors in the induction of G418^r colonies. Six G418^r clones were derived from BALB 3T3, NIH 3T3 and FRSK cells, and cocultured with G418 sensitive (G418^s) parent cells in G418 medium. The colony formation capacity of all G418^r cell clones decreased with the increasing number of plated G418^s cells. Cell-cell contact appeared to be necessary to reduce the colony formation of G418^r cells, and contact-dependent G418^r cell killing was probably not related to gap junction formation. Contact-mediated cell killing is a likely explanation for the observation that induction of G418^r colonies is often reduced under conditions of high-density plating, long treatment of DNA-CaPO₄ precipitates, and long expression time of G418 selection. These results suggest that in some instances transfection efficiency using pSV2-neo DNA should be carefully evaluated because culture conditions can mask the induction of G418^r colonies.     

丁酸钠对杂交瘤细胞增殖及抗体分泌的影响

用含不同浓度丁酸钠完全培养液和无血清培养液培养分泌抗组织型纤溶酶原激活剂(t-PA)单克隆抗体的杂交瘤细胞。当丁酸钠浓度为0.25～0.5 mmol/L时,对杂交瘤细胞分泌抗体能力具有刺激作用,提高抗体效价2～4倍,对细胞生长无抑制作用。表明丁酸钠可作为一种添加剂用以提高杂交瘤细胞分泌抗体的能力。     

Tumourigenic transformation of mouse NIH 3T3 cells by transfection with plasmid pSV2neo

This study reports high incidence tumourigenic activity of the pSV2neo plasmid demonstrated by transfection of NIH 3T3 cells. The plasmid is frequently used as a dominant selectable marker for confirming successful gene transfection, and the findings indicate a need for caution in interpretation and design of assays for oncogenes which use co-transfection and subsequent selection with neomycin.     

Isolation and characterization of a new immortal rat astrocyte with a high expression of NGF mRNA

We have established a new line of immortalized rat astrocytes through transfection of plasmid pSV3-neo encoding the large T antigen of simian virus 40 into normal astrocytes. One of these immortalized astrocytes (ACT-57) with a flat and polygonal cell shape, exhibited stable growth in a chemically defined medium (modified N-2 medium) as well as in medium containing ordinary serum. ACT-57, retained a detectable level of expression of glial fibrillary acidic protein (GFAP) and its mRNA, and exhibited a stronger expression of nerve growth factor (NGF) mRNA than that of normal rat astrocytes or C6 glioma cells. NGF mRNA was significantly up-regulated by phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and gamma-amino-n-butyric acid (GABA) but not by hydrocortisone. None of stimulants (TPA, dibutyryl cyclic AMP (db-cAMP), hydrocortisone, L-glutamate, carbacol, GABA, dopamine, or isoproterenol) changed the expression level of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). There was a discrete difference between ACT-57 and normal astrocytes in the response to GABA and isoproterenol. These findings imply that normal cortical astrocytes possess a functional heterogeneity whereas the clonal astrocyte, ACT-57, does not, indicating that ACT-57 cells may be useful for in vitro studies of neuron-astrocyte interactions involving the induction of neurotrophic factors such as NGF.     

Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen,using linkage of cotransfected markers

We report the molecular cloning of a human gene MER-2located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO ×human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2expression.