动物细胞培养及单克隆抗体

941449重组CHO细胞在其细胞周期中表达外源基因(β-半乳糖苷酶)[英]/Gu,M.B.…∥Biotechnol.Bioeng.-1993,42(9).-1113～1123[译自DBA,1993,12(25),93-14684] 用含二氢叶酸-还原酶基因(dhfr)和编码细菌β-半乳糖苷酶的LacZ基因的表达载体转染CHOβ-G72-16细胞,研究了单个该细胞中的外源蛋白     

牛αS1-酪蛋白5′调控序列驱动人α-LA基因与LacZ基因在牛乳腺上皮细胞中的表达

将牛αS1-酪蛋白5′调控序列约1.2 kb的片段,连接到含SV40启动子调控下β-半乳糖苷酶基因(LacZ)的PSV载体上,做为启动子,在其后连接0.76 kb的人α-乳白蛋白基因(α-LA),构建真核表达载体 αS1-LA-psv．采用组织块接种法,培养奶牛乳腺上皮细胞,经传代纯化后,对细胞接种存活率、群体倍增时间、生长曲线、形态学等生物学性状进行检测,用免疫荧光细胞染色法对培养的奶牛乳腺上皮细胞进行角蛋白18鉴定,结果表明,成功建立奶牛乳腺上皮细胞系,细胞传至20代以上时仍保持旺盛的增殖活力．将构建的真核表达载体 αS1-LA-psv 转染奶牛乳腺上皮细胞,培养24～120 h均检测到了β-半乳糖苷酶的表达;培养72 h检测到细胞中人α-乳白蛋白的表达,表达量约为0.64 g/L．实验结果表明,建立的奶牛乳腺上皮细胞系具有外源基因表达活性,得到的牛αS1-酪蛋白5′调控序列能作为启动子指导外源基因的表达,构建的真核表达载体能在体外培养的牛乳腺上皮细胞中同时表达人α-乳白蛋白和β-半乳糖苷酶．     

牛αS1-酪蛋白5＇调控序列驱动人α-LA基因与LacZ基因在牛乳腺上皮细胞中的表达

将牛αS1-酪蛋白5＇调控序列约1．2kb的片段,连接到含SV40启动子调控下β-半乳糖苷酶基因（LacZ）的PSV载体上,做为启动子,在其后连接0．76kb的人α-乳白蛋白基因（α-LA）,构建真核表达载体αS1-LA-psv．采用组织块接种法,培养奶牛乳腺上皮细胞,经传代纯化后,对细胞接种存活率、群体倍增时间、生长曲线、形态学等生物学性状进行检测,用免疫荧光细胞染色法对培养的奶牛乳腺上皮细胞进行角蛋白18鉴定,结果表明,成功建立奶牛乳腺上皮细胞系,细胞传至20代以上时仍保持旺盛的增殖活力．将构建的真核表达载体αS1-LA-psv转染奶牛乳腺上皮细胞,培养24～120h均检测到了β-半乳糖苷酶的表达;培养72h检测到细胞中人α-乳白蛋白的表达,表达量约为0．64g／L．实验结果表明,建立的奶牛乳腺上皮细胞系具有外源基因表达活性,得到的牛αS1-酪蛋白5＇调控序列能作为启动子指导外源基因的表达,构建的真核表达载体能在体外培养的牛乳腺上皮细胞中同时表达人α-乳白蛋白和β-半乳糖苷酶．     

EGFP-LacZ双报告基因真核表达载体的构建及体外表达

构建(β-半乳糖苷酶与增强型绿色荧光蛋白(EGFP)双报告基因的真核表达载体,并在真核细胞中表达。采用PCR方法从质粒pLenti6/V5-GW/LacZ 中获取LacZ全基因,与pEGFP-C1重组后构建真核表达载体pEGFP-C1-LacZ。该重组质粒经PCR和酶切方法鉴定后,在脂质体介导下转染293FT细胞株及鸡胚成纤维细胞,通过荧光观察和组织化学方法检测Egfp和LacZ基因的表达。结果表明,LacZ基因被克隆到pEGFP-C1,成功构建了双报告基因真核表达载体。该重组质粒转染后的细胞呈现绿色荧光,组织化学方法检测到呈蓝色的细胞,表明两个报告基因均能在细胞内正确表达。     

阳离子脂质体转染人类骨骼肌原代细胞的初步研究

探讨不同脂质体介导基因转染人类骨骼肌原代细胞的转染效率和基因的表达.将含有β-半乳糖苷酶LacZ结构基因的质粒,用三种不同的阳离子脂质体导入人类骨骼肌原代细胞中,通过X-Gal染色观察不同的转染效率.结果发现,Fugene 6转染效率最高,蓝染细胞达10%,其脂质体与DNA的最佳比例为3∶ 2.Fugene 6可有效地将外源基因导入骨骼肌原代细胞,而且外源基因可以长效高效地表达,有望用来作为基因治疗的载体.     

大肠杆菌启动基因的重组和表达 Ⅰ.不同启动基因对β-半乳糖苷酶结构基因表达的影响

质粒pKL6带有β-半乳糖苷酶结构基因(lacZ),启动基因突变失活,lacZ不能表达。在lacZ前插入外源DNA片段,观察lacZ的表达,可以研究启动基因的功能作用。我们用大肠杆菌染色体DNA的HindⅢ片段与pKL6重组,获得一批重组体,对这些重组体的β-半乳糖苷酶的活力水平和限制性图谱作了分析,对它们用作表达载体的可能性进行了讨论。     

牛αS1-酪蛋白5'调控序列驱动人α-LA基因与LacZ基因在牛乳腺上皮细胞中的表达

将牛αS1-酪蛋白5′调控序列约1.2 kb的片段,连接到含SV40启动子调控下β-半乳糖苷酶基因(LaeZ)的PSV载体上,做为肩动子,在其后连接0.76kb的人仅α-乳白蛋白基凼(α-LA),构建真核表达载体αS1-LA-psv.采用组织块接种法,培养奶牛乳腺上皮细胞,经传代纯化后,对细胞接种存活率、群体倍增时间、生长曲线、形态学等生物学性状进行检测,用免疫荧光细胞染色法对培养的奶牛乳腺上皮细胞进行角蛋白18鉴定,结果表明,成功建立奶牛乳腺上皮细胞系,细胞传至20代以上时仍保持旺盛的增殖活力.将构建的真核表达载体αS1-LA-psv转染奶牛乳腺上皮细胞,培养24～120h均检测到了β-半乳糖苷酶的表达;培养72 h检测到细胞中人α-乳白蛋白的表达,表达量约为0.64 g/L.实验结果表明,建它的奶牛乳腺上皮细胞系具有外源基因表达活性,得到的牛αS1-酪蛋白5′调控序列能作为启动子指导外源基因的表达,构建的真核表达载体能在体外培养的牛乳腺上皮细胞中同时表达人α-乳白蛋白和β-半乳糖苷酶.     

分子无性繁殖载体的组建——Ⅰ.乳糖操纵子的重组和表达

用包含乳糖操纵子的λplac 5 EcoR1 DNA片段与质粒pBR 322重组,获得一个重组质粒pMG4。几种限制性内切酶的分析结果表明,pMG4上乳糖操纵子(lac)和抗氨基苄青霉素基因的转录方向一致;Iac插入片段λ区的Hind Ⅲ切点和pBR 322抗四环素基因上的Hind Ⅲ切点相邻近。用pMG4 Hind Ⅲ片段转化大肠杆菌C_(600),筛选Ap~rTc~s lac~ 转化体,得到另一个lac重组质粒pMG401,在β-半乳糖苷酶结构基因近羧基端有一个EcoR1切点,插入外源基因可以置于lac控制下而实现表达。同时,我们还测定了包含pMG4、pMG401或λplac 5不同细胞的β-半乳糖苷酶活力,对lac的表达作了分析。     

分子无性繁殖载体的组建——Ⅰ.乳糖操纵子的重组和表达

用包含乳糖操纵子的λplac 5 EcoR1 DNA 片段与质粒pBR 322重组,获得一个重组质粒pMG4。几种限制性内切酶的分析结果表明,pMG4上乳糖操纵子(lac)和抗氨基苄青霉素基因的转录方向一致;Iac 插入片段λ区的Hind Ⅲ切点和pBR 322抗四环素基因上的Hind Ⅲ切点相邻近。用pMG4 Hind Ⅲ片段转化大肠杆菌C_(600),筛选Ap~r Tc~s lac~ 转化体,得到另一个lac 重组质粒pMG401,在β-半乳糖苷酶结构基因近羧基端有一个EcoR1切点,插入外源基因可以置于lac 控制下而实现表达。同时,我们还测定了包含pMG4、pMG401或λplac 5不同细胞的β-半乳糖苷酶活力,对lac 的表达作了分析。     

假单胞菌M18转录调节因子σs基因与无启动子lacZ基因融合突变株的构建和鉴定

通过菌落原位杂交和Southern杂交,从假单胞菌M18基因组文库中克隆了rpoS基因及相邻序列。为了深入研究影响rpoS基因表达的调控因素,运用同源重组技术,将无启动子β-半乳糖苷酶基因(-′lacZ)插入并融合于rpoS基因中,构建了假单胞菌M18rpoS基因突变株M18SZ。Miller法测定显示,突变株M18SZ的β-半乳糖苷酶可高达480U,而野生株检测不到β-半乳糖苷酶活性。表明,突变株中的rpoS基因与无启动子β-半乳糖苷酶基因已融合并且表达。在KMB培养基中生长量测定(OD600)的结果表明,突变株与野生株生长存在显著差异。     

Generation of stable cell line by using chitosan as gene delivery system

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.     

Expression in vivo of the lacZ gene, delivered to mouse lungs using synthetic microspheres

A capacity of MF-2 synthetic microspheres to serve as the vehicle for transfer of the marker LacZ gene to mouse lung epithelial cells was studied after a single intranasal administration of the MF-2/gene complex. Two types of plasmids carrying marker gene LacZ were used in the experiments: with cytoplasmic (pCMV-LacZ) and nuclear (pCMV-nlsLacZ) localization of the gene product (beta-galactosidase). As early as 7 days after the complexes MF-2/pCMV-LacZ and MF-2/pCMV-nlsLacZ were administered, specific staining for beta-galactosidase revealed this enzyme activity in the epithelial cells of bronchi, bronchioli, and alveoli. The maximum in vivo of the marker gene in the MF-2/pCMV-LacZ complex was observed at day 14 to 21 after administration and the corresponding gene product was detected during the following two months. The MF-2-mediated gene transfer led to a twofold increase in beta-galactosidase activity relative to the case when the "unbound" pCMV-LacZ plasmid was administered. These results suggest that the synthetic microsphere-mediated transfer of alien genes to the lung of experimental animals is promising. Microspheres may be used in gene therapy for pulmonary affections, in particular cystic fibrosis.     

Stable transformation of mouse teratocarcinoma stem cells with the dominant selective marker Eco.gpt and retention of their developmental potentialities

Transformation of PCC4 mouse teratocarcinoma stem cells was obtained using a dominant selective marker, the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT), coded by the bacterial Eco.gpt gene placed under the control of the early SV40 genes in the vector pSV2gpt. An average of 20 colonies of transformed cells was obtained, using the calcium phosphate technique, 10 microg DNA vector, no carrier DNA and 1 x 10(6) recipient cells. Five independent Eco.gpt-transformed PCC4 cell lines were propagated in selective medium and assayed for XGPRT activity. All of them had the ability to convert [14C]xanthine to xanthine monophosphate. pSV2gpt sequences were present and associated with high mol. wt. cellular DNA. pSV2gpt sequences and XGPRT activity were both conserved in the three clones that were propagated in non-selective medium for 30 generations. The transformed PCC4 cells retained their ability to produce, in host mice, teratocarcinoma tumors composed of embryonal carcinoma and various differentiated tissues. Thus, pSV2gpt can be used as a dominant marker to select teratocarcinoma stem cells co-transformed with genes that are not selectable by themselves.     

Nonsense RNA: a tool for specifically inhibiting the expression of a gene in vivo

We describe a general technique to inhibit gene expression in eukaryotic cells. The gene we chose to inhibit was the E. coli LacZ gene (encoding beta-galactosidase), which has previously been cloned into a eukaryotic expression vector [1]. This plasmid is called pCH110. We constructed a variant of pCH110 in which we flipped a 2566 base pair 5' fragment of the LacZ gene into the antiparallel orientation. The plasmid containing this mutated LacZ gene is called pNSLacZ (NS signifies non-sense coding sequence). When equal amounts of pCH110 and pNSLacZ are co-transfected into 3T6 mouse fibroblasts, the beta-galactosidase activity is decreased by approximately a factor of ten. Increasing the ratio of pNSLacZ to pCH110 above 1:1 does not appreciably increase the level of inhibition. Next, we prove the specificity of the inhibition by adding a third gene to the transfection mixture. For this purpose, we used pSVneo beta, a plasmid which expresses a phosphotransferase. We found that even when the beta-galactosidase activity was diminished by a factor of 10, the phosphotransferase activity was unaffected. Therefore, we have demonstrated that: the presence of an antiparallel copy of the LacZ gene results in a significant and specific diminution of the LacZ gene's expression; only a fraction of the LacZ gene needs to be in the antiparallel orientation in order to observe this effect. These results suggest that this technique can serve as a tool to decrease the level of gene expression in order to study the function of specific genes, or as a therapeutic manoeuvre in the treatment of disorders of abnormal gene expression.     

LacZ transgenic mice and immunoelectron microscopy: an ultrastructural method for dual localization of beta-galactosidase and horseradish peroxidase.

Transgenic animals bearing the reporter gene, LacZ, encoding the histochemical enzyme, beta-galactosidase, are increasingly becoming available. Similarly, antibody conjugates consisting of specific IgGs coupled to horseradish peroxidase (HRP) are widely used for Western blotting, ELISA, and immunohistochemistry. Here we provide a detailed fixation and histochemical protocol for the simultaneous electron microscopic visualization and discrimination of beta-galactosidase and peroxidase reaction products within mouse kidney. After incubation of transgenic LacZ tissues with IgG-HRP conjugates, samples were lightly fixed with 2% paraformaldehyde and 0.4% glutaraldehyde and processed for peroxidase histochemistry. Tissues underwent beta-galactosidase histochemistry, were refixed with glutaraldehyde, osmicated, and embedded. In Flk1/LacZ mice, we immunolocalized anti-laminin beta1 chain IgG-HRP specifically to developing glomerular basement membranes, whereas Flk1/LacZ was expressed only by glomerular endothelial cells. In Epas1/LacZ mice, we immunolocalized anti-platelet endothelial cell adhesion molecule-1 specifically to glomerular endothelial plasma membranes, whereas Epas1/LacZ was expressed by both glomerular endothelial and mesangial cells. This dual ultrastructural localization technique should be broadly applicable for immunoelectron microscopic studies in LacZ transgenic animals, particularly those where LacZ expression and antibody-HRP binding are both relatively abundant.     

Synthesis of radioiodine-labeled 2-phenylethyl 1-thio-beta-D-galactopyranoside for imaging of LacZ gene expression

A potent inhibitor of beta-galactosidase (EC 3.2.1.23), 2-phenylethyl 1-thio-beta-D-galactopyranoside (PETG), was radioiodinated for noninvasive imaging of LacZ gene expression. In order to introduce radioiodine to the phenyl ring of PETG, 2-(4-bromophenyl)ethanethiol was prepared and attached to the C-1 position of beta-D-galactose pentaacetate under conditions that resulted in the exclusive formation of the beta anomer. The bromo group of PETG was converted to the tributylstannyl group where radioiododemetallation was carried out. Radioiodine-labeled PETG tetraacetate was purified by HPLC, which can be used as a prodrug for biological evaluation or hydrolyzed to 2-(4-[123I/125I]iodophenyl)ethyl 1-thio-beta-D-galactopyranoside ([123I/125I]7) under basic conditions. The resulting radioiodine-labeled PETG was obtained in overall 62% radiochemical yield (decay-corrected) and with specific activity of 46-74 GBq/micromol.     

降低PARP酶活性对外源基因稳定整合的影响

通过使用 PARP酶的抑制剂研究了降低PARP酶的活性对外源基因转染效率及整合作用的影响, 结果表明,转染的外源基因的吸收及瞬时表达不依赖于PARP酶的活性,而外源基因的整合作用与PARP酶的活性有关。 Abstract:In this study,the effect of lowering PARP activity on the transfection of cultured cells with foreigh genes was evaluated.The results indicated that PARP enzyme involved in the stable integration of foreign genes into the host genome.However,inhibition of PARP activity exhibited no effect on both the uptake into the cells and the expression of the forging genes.     

A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase

Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.