稳定、高效表达肝细胞生长因子的中国仓鼠卵巢细胞系的构建

目的:构建hHGF-pCDNA3.1表达载体,在中国仓鼠卵巢细胞中获得具有生物学活性的重组人肝细胞生长因子蛋白的高效、稳定表达。方法:RT-PCR扩增人肝脏中hHGF全长cDNA片段,克隆入pCDNA3.1( )真核表达载体中,构建hHGF-pCD-NA3.1重组质粒并转染中国仓鼠卵巢细胞,经筛选得到了高效、稳定表达hHGF的细胞克隆,采用RT-PCR和ELISA方法检测hHGF在中国仓鼠卵巢细胞中的表达。结果:酶切及测序结果表明重组质粒构建正确,RT-PCR显示细胞的rhHGF mRNA呈现高水平,ELISA检测hHGF在细胞中的分泌性表达,浓度达10ug/L。结论:成功地在中国仓鼠卵巢细胞中获得了hHGF蛋白的高效、稳定表达。为下一步将表达hHGF的细胞微囊化制备基因工程细胞,并移植用于相关疾病的基因治疗奠定了基础。     

二氢叶酸还原酶缺陷型中华仓鼠卵巢细胞偏爱密码子的研究

目的研究二氢叶酸还原酶缺陷型中华仓鼠卵巢细胞（CHO dhfr-）的偏爱密码子。方法建立CHO dhfr-细胞高丰度mRNA的cDNA文库，对该文库进行签定以获得合格的编码蛋白质的cDNA序列，统计密码子使用频率并与CUTG数据库中的中华仓鼠比较，同时对所获得的cDNA序列进行对应统计分析。结果获得了50个合格的CHO dhfr-细胞高丰度蛋白的cDNA序列，与中华仓鼠密码子使用频率比较，除了Pro，Arg外，其余氨基酸的最高使用频率的密码子在CHO dhfr-细胞和中华仓鼠中都相同。同时根据对应分析确定了能解释最多变量（14．7％）的第一主成分，确定了22个同义密码子为CHO dhfr-细胞偏爱密码子。结论CHO dhfr-细胞存在自身偏爱的密码子，提示密码子的偏爱性是不同哺乳动物细胞功能差异的因素之一，也为外源基因在CHO dhfr-细胞中通过优化密码子而获得高表达提供了新思路。     

Valinomycin-induced apoptosis in Chinese hamster ovary cells

Accumulating evidence endorses that excessive K(+) efflux is an ionic mechanism underlying apoptosis both in neuronal and non-neuronal cells. K(+) channels play important roles in mediating the pro-apoptotic K(+) efflux. Chinese hamster ovary (CHO) cells have been widely used for gene transfection experiments. These cells lack detectable endogenous voltage-gated K(+) channels. We were interested in knowing whether the absence of endogenous K(+) channels would render wild-type CHO cells more resistant to apoptotic death. We also wished to determine if direct stimulation of K(+) efflux would trigger apoptosis in these cells. Exposing CHO cells to hypoxia (1% O(2)) or to a typical apoptotic insult of serum deprivation for up to 24h did not affect cell survival. On the other hand, the K(+) ionophore valinomycin caused substantial cell death within 12h of its application. Valinomycin-treated CHO cells underwent several apoptotic events, including phosphatidylserine (PS) membrane translocation, caspase-3 activation, and mitochondrial membrane depolarization during the first few hours of exposure. Reducing K(+) efflux by elevating extracellular K(+) concentrations noticeably attenuated valinomycin-induced cell death. This study reinforces a K(+) efflux-mediated apoptotic mechanism in CHO cells and may help to explain the unique feature of their higher tolerance to apoptosis.     

Statistical analyses for in vitro cytogenetic assays using Chinese hamster ovary cells

It is a widely held view that objective statistical criteria are needed for the evaluation of genetic toxicity assays. This paper presents statistical methods for the analysis of data from in vitro sister chromatid exchange (SCE) and chromosome aberration tests that use Chinese hamster ovary cells. For SCEs, an extensive study of solvent control results demonstrated that there is a substantial interday component of variability in the data, and that a Poisson sampling model is applicable to data generated via the protocol of Galloway et al [1985]. Consequently, a trend test for evidence of a dose response is proposed for such SCE data. As an illustration of this statistical method, analysis of data previously considered to be negative [Gulati et al, 1985] indicates that di(2-ethyl-hexyl) phthalate induces a weak, but reproducible, SCE dose response in CHO cells. Monte Carlo methods are used to show that the trend test is more sensitive than four other statistical procedures considered for the analysis of Poisson-distributed SCEs. A similar trend test for dose response in proportions is proposed for chromosome aberration data, where the percent of cells with chromosome aberrations is the response of interest. Sensitivity (or power) studies indicate that three doses and a control with 50 cells/dose point is a reasonable design for an in vitro SCE study that uses the Galloway et al protocol. For in vitro chromosome aberrations, however, three doses and a control with 100 cells/dose point appears to produce too insensitive an assay; an increase to 200 cells/dose point in the Galloway et al protocol seems worthy of serious consideration.     

Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes

The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. CFTR expression is controlled temporally and cell specifically by mechanisms that are poorly understood. Insight into CFTR regulation could be facilitated by the successful introduction of the entire 230 kb human CFTR and adjacent sequences into mammalian cells. To this end, we have introduced two different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells. Clonal cell lines containing human CFTR were identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described. Integration of the human CFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescence in situ hybridization (FISH). Southern blot analysis suggested that on the order of one copy of human CFTR was integrated per CHO cell genome. Fiber-FISH and restriction analysis suggested that CFTR remained grossly intact. Northern analysis showed full-length, human CFTR mRNA. Immunoprecipitation followed by phosphorylation with protein kinase demonstrated mature, glycosylated CFTR. Finally, chloride secretion in response to cAMP indicated the functional nature of the human CFTR. This study provides several novel results including: (i) functional human CFTR can be expressed from these YACs; (ii) CHO cells are a permissive environment for expression of human CFTR; (iii) the level of human CFTR expression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH of CFTR integrity correlates with functional gene expression. These YACs and the cell lines derived from them should be useful tools for the study of CFTR expression.      

Efficient rescue of measles virus from cloned cDNA using SLAM-expressing Chinese hamster ovary cells

We here report a highly efficient reverse genetics system for measles virus (MeV), using Chinese hamster ovary cells constitutively expressing a MeV receptor human signaling lymphocyte activation molecule (CHO/hSLAM cells). The recombinant vaccinia virus vTF7-3 that encodes the T7 RNA polymerase under the control of the early/late promoter was used in the system. Replication of vTF7-3 was highly restricted in CHO/hSLAM cells, but the virus could still drive the T7 promoter, allowing us to recover MeV from the transfected cDNA efficiently. With this system the number of infectious centers, in which MeV replication cycles are initiated from transfected cDNAs, was approximately 100 times higher than that with the previous system (. J. Virol. 74, 6643-6647), and the recovery rate was 100%. The wild-type MeV that encodes the lac-Z gene of approximately 3.2kb in length, was easily generated with this CHO/hSLAM system, while such virus could not be recovered with the previous system. Since SLAM acts as a cellular receptor for both MeV vaccine and wild-type strains, the Edmonston vaccine strain was also recovered with this system more efficiently than with any other systems reported previously. Thus, the CHO/hSLAM-based system would expand applications of the MeV reverse genetics by allowing productions of mutant MeVs that have been difficult to generate with less efficient systems.     

Synthesis of prostaglandins in cholera toxin-treated Chinese hamster ovary cells

The prostaglandin (PG) and adenosine 3',5'-cyclic monophosphate (cAMP) responses of Chinese hamster ovary (CHO) cells were measured after cholera toxin (CT) exposure to evaluate dose and kinetic relationships. Release of prostaglandin E2 (PGE2) and the accumulation of cAMP were dependent on the dose of CT, with an effective dose of approximately 10-100 ng/ml within 4 h; the PGE2 response was about four- to six-fold more than that of PGE1. CHO cells exposed to CT also released increased amounts of thromboxane B2 (TxB2), PGF2 gamma, and 6-keto PGF1 gamma (a non-enzymatic degradation product of prostacyclin). Kinetic analysis of CT-treated cells revealed that small peaks of cAMP accumulation and of PGE1 and PGE2 release were detected at approximately 30 min, but larger, progressive PG and cAMP responses were measured 2-4 h later. Exposure of the cells to relatively high doses of membrane-permeable derivatives of cAMP (1 mM) and forskolin (10 microM) caused PGE2 release. Concomitantly, exogenous PGE2 (100 microM) increased intracellular levels of cAMP. We have considered the interrelationship of the cyclo-oxygenase and the cyclic nucleotide pathways relative to the molecular mechanism of CT.     

Targeting vector configuration and method of gene transfer influence targeted correction of theAPRT gene in Chinese hamster ovary cells

A 21-bp deletion in the third exon of theAPRT gene in Chinese hamster ovary (CHO) cells was corrected by transfection with a plasmid containing hamsterAPRT sequences. Targeted correction frequencies in the range of 0.3–3.0×10^–6 were obtained with a vector containing 3.2 kb ofAPRT sequence homology. To examine the influence of vector configuration on targeted gene correction, a double-strand break was introduced at one of two positions in the vector prior to transfection by calcium phosphate-DNA coprecipitation or electroporation. A double-strand break in the region ofAPRT homology contained in the vector produced an insertion-type vector, while placement of the break just outside the region of homology produced a replacement-type vector. Gene targeting with both linear vector configurations yielded equivalent ratios of targeted recombinants to nontargeted vector integrants; however, targeting with the two different vector configurations resulted in different distributions of targeted recombination products. Analysis of 66 independent APRT⁺ recombinant clones by Southern hybridization showed that targeting with the vector in a replacement-type configuration yielded fewer targeted integrants and more target gene convertants than did the integration vector configuration. Targeted recombination was about fivefold more efficient with electroporation than with calcium phosphate-DNA coprecipitation; however, both gene transfer methods produced similar distributions of targeted recombinants, which depended only on targeting vector configuration. Our results demonstrate that insertion-type and replacement-type gene targeting vectors produce similar overall targeting frequencies in gene correction experiments, but that vector configuration can significantly influence the yield of particular recombinant types.     

Isolation and preliminary characterization of Sindbis virus-resistant Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells resistant to the cytolytic effects of Sindbis virus infection have been selected in one step from mutagenized wild-type cells. The mutants were present at frequencies as high as 8.8 × 10^?4, and were obtained virus free following the incubation of the isolates in the presence of anti-Sindbis virus antibody. The mutant phenotypes behaved recessively in somatic cell hybrids, and there were at least two complementation groups among the isolates tested. In addition to the virus-resistant phenotype, the mutants clearly have surface changes as demonstrated by their altered morphology, increased sensitivity to phytohemagglutinin, and increased resistance to diphtheria toxin. Analyses of revertants from one mutant indicated that all of the phenotypes of the mutant resulted from a single genetic alteration. All isolates were partially defective in viral adsorption. Comparison of viral replication in WT and one mutant cell strain suggested that the major effect on viral replication in the mutant studied is at the level of viral mRNA translation.     

Legionella pneumophila inhibits protein synthesis in Chinese hamster ovary cells.

Legionella pneumophila is a gram-negative facultative intracellular parasite that causes Legionnaires disease. To explore the interactions between L. pneumophila and host cells, we have developed a continuous cell line model of infection. We show that about 80% of Chinese hamster ovary (CHO) cells were associated with L. pneumophila after incubation for 3 h at a multiplicity of infection of 20 bacteria per cell. Within 3 to 4 h of incubation with L. pneumophila, protein synthesis of CHO cells was markedly inhibited, as shown by the reduction of incorporation of radiolabeled amino acids into proteins. L. pneumophila did not inhibit transport of amino acids or cause degradation of newly synthesized proteins in CHO cells. Cytochalasin D blocked internalization of L. pneumophila by CHO cells, yet CHO cell protein synthesis was inhibited. These results indicated that L. pneumophila could inhibit host protein synthesis from the cell exterior. L. pneumophila that had been killed with antibiotics prior to incubation with CHO cells still inhibited protein synthesis, indicating that the inhibition of CHO cell protein synthesis occurred in the absence of de novo protein synthesis by L. pneumophila.     

Characterization of the diphtheria toxin-resistance system in Chinese hamster ovary cells

Variations in two general classes of diphtheria toxin-resistant mutants which may be selected from Chinese hamster ovary (CH0-K1) cells and the conditions for their selection are described. The resistance of class I mutants can be overcome with increasing concentrations of toxin. Their entire complement of EF-2 is susceptible to ADP-ribosylation by toxin. Class I includes those strains in which resistance resides at the level of the plasma membrane. The resistance of class II, translational, mutants cannot be overcome by high concentrations of toxin, as all, or a portion, of their EF-2 is insensitive to the action of diphtheria toxin and Pseudomonas exotoxin A. Adjustment of the concentration of toxin used to select resistant mutants can be used to regulate the class of mutant recovered. Metabolic cooperation between cells does not affect recovery of either class I or class II mutants. Resistance is stable in class I strains, but class IIb strains, which possess 50 % resistant and 50 % sensitive EF-2, display a transient high level of resistance which is retained for varying lengths of time following exposure to toxin. Class IIa strains, which possess 100% resistant EF-2, grow normally in saturating concentrations of toxin, but class IIb strains grow at a reduced rate. Evidence is presented which suggests that the gene for EF-2 is functionally diploid in CH0-K1 cells.     

Characterization of human folylpolyglutamate synthetase expressed in Chinese hamster ovary cells

Chinese hamster AUX B1 cells lack the enzyme folylpolyglutamate synthetase (FPGS) responsible for adding polyglutamates to folie acid. The human gene for FPGS was introduced into AUX B1 cells by cotransfection with human genomic DNA and either pSV2neo or pSV2gpt plasmid DNA. Cells were coselected for FPGS expression by growth in the absence of glycine, adenosine, and thymidine, and for drug resistance by growth in geneticin or mycophenolic acid. The presence of human enzyme in transfected cells was identified by relative enzyme activity determinations with ATP or dATP as substrates. The human HeLa cell enzyme displayed a 50% higher activity with dATP, and the hamster enzyme showed a 50% higher activity with ATP. Hamster and human enzymes also differed in the chain length of polyglutamates on cellular folate. Only monoglutamates were detected in AUX B1 cells, while wild-type hamster cells had predominantly hexa- and heptaglutamates, and human HeLa cells had predominantly hepta- and octaglutamates. Transformants with FPGS activity that showed a human enzyme preference for dATP also had folate polyglutamate chain lengths characteristic of the human enzyme.     

Chromosomal instability in an oxygen-tolerant variant of Chinese hamster ovary cells

Background levels of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined in CHO-99 cells, an oxygen-tolerant variant substrain of Chinese hamster ovary (CHO-20) cells capable of stable proliferation under an atmosphere of 99% O₂/1% CO₂, a level of hyperoxia at which cultured mammalian cells normally cannot survive. The mean chromosomal aberration frequency in CHO-99 cells was as high as 1 aberration per cell (mainly chromatid and chromosome gaps and breaks) versus 0.05 aberration/cell in CHO-20 cells, while the SCE frequency was 1.7- to 2.1-fold increased. While most aberrations were apparently distributed at random over the chromosomes, up to 31% of the aberrations appeared to be involved in site-specific fragility at a homologous site in chromosomes Z3 and Z4. Immediately upon shifting CHO-99 cells to air-equilibrated conditions their SCE frequency decreased to the control level, whereas the aberration rate persisted at a still elevated level of 0.16–0.31 aberration per cell, even after a culture period of 14 weeks under normoxia. This indicates that at least part of the chromosomal instability is a constitutional property of the variant cells, i.e., not directly dependent upon hyperoxic stress. In CHO-99 × CHO-20 hybrids the occurrence of chromatid-type aberrations and fragile site but not that of chromosome-type aberrations was suppressed under normoxic conditions, suggesting that chromatid-type aberrations and fragile site expression on the one hand and chromosome-type aberrations on the other hand are mediated by different constitutional defects in CHO-99 cells. No gross alterations in (deoxy)ribonucleoside triphosphate pools were detected in CHO-99 cells that could be held responsible for their chromosomal instability. In addition, no increased level of DNA damage was detected by the technique of alkaline elution. The excessive chromosomal instability in CHO-99 cells, as observed under hyperoxic conditions, may originate from reactive intermediates giving rise to DNA double-strand breaks and/or a type of DNA lesion that is resistant to the conditions of the alkaline elution technique. However, alternative mechanisms based upon reactive species interfering with DNA replication/repair processes cannot be excluded.     

Codominant translational mutants of Chinese hamster ovary cells selected with diphtheria toxin

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a universal hybridizer (10).     

大鼠重组骨桥蛋白在中国仓鼠卵巢细胞中的稳定表达和纯化

目的 构建包含大鼠骨桥蛋白(OPN)的真核表达载体,获得稳定表达重组蛋白的中国仓鼠卵巢(CHO)细胞株并对蛋白进行纯化,为OPN在大鼠模型实验中的进一步功能学研究奠定基础.方法 通过脂质体lipofectamine 2000将重组质粒pLVX-OPN转染到CHO细胞中,嘌呤霉素筛选出稳定克隆细胞.Real-time PCR法检测转染后CHO细胞中OPN的mRNA表达水平,酶联免疫吸附实验法(ELISA)检测上清中OPN分泌水平.取CHO细胞72 h无血清培养上清进行镍柱亲和层析纯化,ELISA法和Western blot法分别检测产量和纯度.结果 pLVX-OPN表达载体经DNA琼脂糖电泳鉴定为阳性,且质粒测序成功.经质粒pLVX-OPN转染的CHO细胞中OPN mRNA表达水平是转染前的上千倍,上清分泌蛋白水平较转染前增加.上清分泌的OPN蛋白通过镍柱亲和层析纯化后纯度有所提高,产量在30％以上.结论 pLVX-OPN真核表达载体的成功构建和在CHO细胞中的稳定表达及纯化为OPN的功能学研究提供了有效工具.     

Vitronectin mediates internalization of Neisseria gonorrhoeae by Chinese hamster ovary cells.

Gonococci producing a distinct opacity protein (OpaA in strain MS11) adhere to and are efficiently internalized by cultured epithelial cells such as the Chang conjunctiva cell line. Both adherence and uptake require interactions between OpaA and heparan sulfate proteoglycans on the mammalian cell surface. Chinese hamster ovary (CHO) cells also support adherence of gonococci through interactions of OpaA with cell surface heparan sulfate proteoglycans. However, despite this similarity in the requirements for adherence, CHO cells are not capable of internalizing gonococci. In this report, we characterized this apparent deficiency and identified a factor in fetal calf serum (FCS) which is capable of mediating uptake of gonococci by CHO cells. In the absence of FCS, OpaA+ gonococci adhered to but were not internalized by CHO cells, whereas in the presence of up to 15% FCS, the bacteria were efficiently internalized by the cells. Preincubation of bacteria, but not cells, with FCS also stimulated internalization, suggesting that a factor present in FCS was binding to the surface of gonococci and subsequently stimulating entry. Using a combination of chromatographic purification procedures, we identified the adhesive glycoprotein vitronectin as the serum factor which mediates the internalization of gonococci by CHO cells. Vitronectin-depleted serum did not support gonococcal entry, and this deficiency was restored by the addition of purified vitronectin. Further experiments using a set of gonococcal recombinants, each expressing a single member of the family of Opa outer membrane proteins, demonstrated that vitronectin bound to the surface of OpaA-producing gonococci only and that the vitronectin-mediated uptake by the CHO cells was limited to this bacterial phenotype. To our knowledge, our data are the first example that vitronectin can serve as a molecule that drives bacterial entry into epithelial cells.