GFP质控芯片的初步研究

目的：建立一种质量控制芯片来监测样品标记、杂交和检测过程中的失误。方法：针对GFP基因设计的4条60mer寡核苷酸探针和1条阳性对照探针polv（U）与流感寡核苷酸探针一起打印在DAKO玻片上,并构建了GFP基因的克隆载体和体外表达载体,将从这两种重组载体上获得的绿色荧光蛋白（Green Fluorescent Protein,GFP）基因的ILNA、DNA片段和人的全血样品中的DNA用限制性显示技术（Restriction Display technology,RD）扩增标记,将标记的样品和荧光标记的通用引物U分别与芯片杂交、检测,并对扫描的结果进行统计分析。结果：GFP探针与相应的样品杂交时出现阳性信号,阳性对照探针在所有的杂交中均出现阳性信号,而空白对照则未检测荧光信号。结论：建立的质控芯片具有较好的敏感性和特异性,可以用于基因芯片中的质量监控。     

绿色木霉纤维素酶CBHII基因的分子克隆

本文以噬菌体lambda EMBL3 DNA为载体,通过克隆绿色木酶(Trichoderma viride)高分子量基因组DNA的部分酶解片段,并将重组分子进行体外包装后侵染Escherichia.coli K802,由此构建了绿色木霉基因文库。以李氏木霉(Trichoderma reesei)纤维素酶CBHII基因的末端片段为探针,用轮迥噬菌斑原位杂交从文库中筛选出CBHII基因的阳性克隆5个,随机取其中3个克隆用上述探针作斑点杂交,结果进一步证明克隆了全长或近全长的绿色木霉CBHII基因,用李氏木霉CBHI基因的末端片段探针作斑点杂交,结果提示CBHI与CBHII基因的末端序列之间无同源性存在。从斑点杂交的阳性克隆中提取DNA,酶切鉴定插入片段的长度,并克隆于质粒pUC19,Southern杂交结果证明获得了含绿色木霉CBHII基因的重组质粒pCBHII-14。     

5-Aza-CdR对人胃癌细胞株生物学行为及RASSFlA mRNA表达的影响

观察不同浓度的5-氮-2′-脱氧胞苷(5-Aza-CdR)对人胃癌细胞株BGC-823、SGC-7901、MKN-28生长及RASSF1A mRNA表达的影响。方法:分别以0．4μmol/L、1．6μmol/L、6．4μmol/L、25．6μmol/L、102．4μmol/L浓度的5-Aza-CdR处理人胃癌细胞株BGC-823、SGC-7901、MKN-28,MTT比色法测定72h时间段的吸光度值、计算抑制率,流式细胞仪检测5-Aza-CdR对胃癌细胞株生长周期及凋亡的影响,RT-PCR检测5-Aza-CdR处理前、后抑癌基因RASSF1A mRNA的表达。结果:5-Aza-CdR抑制体外培养人胃癌细胞株BGC-823、SGC-7901、MKN-28生长,呈浓度依赖性;5-Aza-CdR能有效诱导BGC-823、SGC-7901、MKN-28细胞凋亡;RT-PCR检测人胃癌细胞株SGC-7901、MKN-28无RASSF1A mRNA表达,经5-Aza-CdR处理后基因重新表达, BGC-823处理前后RASSF1A mRNA均有表达。结论:新型抑癌基因RASSF1A与胃癌的发生相关,5-Aza-CdR能抑制胃癌细胞株的增殖,并促进凋亡,其机制可能与RASSF1A基因的重新表达有关。     

胃癌823细胞cDNA文库的建立

随着DNA重组技术的发展,某些与细胞癌变有关的基因的分离与克隆越来越受到人们的重视。要达到分离和纯化癌基因及其产物的目的,一个较为有效的方法就是构建cDNA文库。最近我们建立了胃癌823细胞(BGC-823)的cDNA文库,希望能从文库中分离到与胃癌     

p53基因对人胃癌细胞系恶性生长的影响

以p53cDNA为探针,用Southern印迹法对人胃癌细胞系BGC823进行了检测,发现该细胞中P53基因存在异常．将可在真核细胞表达的重组野生型P53质粒PC53一SN3和突变型P53质粒PC53—SCX3,用脂质体介导法,分别导入BGC823细胞,获得了较长时间耐受G418的多个阳性克隆．Southern印迹法证实阳性克隆细胞中有外源性P53基因存在．比较BGC823细胞,转染野生型及突变型P53质粒的3种细胞生长曲线和软琼脂集落形成状况发现,野生型P53基因对BGC823细胞恶性生长有一定抑制作用．     

靶向survivin的siRNA对胃癌BGC-823细胞增殖和转移能力的影响

目的探讨沉默生存素（survivin）基因表达的干扰RNA对人胃癌BGC-823细胞增殖和成瘤能力的影响。方法应用已经在细胞上验证能够有效沉默survivin的小分子干扰RNA（shRNA-survivin-1）,并在体外实验的基础上,建立稳定表达干扰RNA细胞系,进一步探讨干扰RNA稳定表达对胃癌BGC-823细胞生长和裸鼠移植成瘤的影响。结果 shRNA-survivin-1有效沉默人胃癌BGC-823细胞survivin mRNA的表达,成功筛选shRNA-sur-vivin-1稳定表达细胞株BGC/siRNA-1细胞,实验表明,BGC/siRNA-1细胞的生长曲线缓慢上升,细胞增殖能力下降;BGC/siRNA-1细胞裸鼠移植成瘤体积与对照组相比,明显减小（P〈0.05）。结论 shRNA-survivin-1可以沉默survivin基因的表达,可以显著抑制胃癌BGC-823细胞的增殖,并降低胃癌BGC-823细胞的成瘤能力,本研究为靶向survivin的RNA干扰在胃癌的基因治疗提供了有力的理论依据和技术储备。 更多还原     

生物素标记的6种cDNA探针检测S180及其克隆细胞株mRNA的表达

目的检测S180及其克隆细胞株S1B11及S2D9mRNA的表达,对这些细胞株进行识别和质量控制.方法用生物素标记的6种cDNA探针,细胞玻片原位杂交的方法检测细胞中mRNA的表达.结果北京市肿瘤研究所(肿瘤所)保存的S180与生物素标记的P16、c-fos、c-myc及c-jun探针杂交阳性,克隆细胞株S1B11与c-fos及c-jun探针杂交阳性,克隆细胞株S2D9与c-fos、c-myc及c-jun探针杂交阳性.结论肿瘤所S180及其2株克隆细胞中mRNA的表达不同,c-myc基因的表达与否可以把S1B11及S2D9克隆细胞区别开;细胞株致瘤性与癌基因表达有关.     

逆转录病毒载体介导入GM-CSF基因转染肿瘤细胞

本文应用逆转录病毒载体pZIP Neo SV(X)介导人GM-CSF基因转染肿瘤细胞获得表达。经Lipofectin将含有人GM-GSF基因的重组逆转录病毒载体pZIP-GM转染兼性病毒包装细胞系PA317,继之用病毒收获液感染人肝癌细胞SMMC7721和人胃癌细胞BGC-823,经GM-CSF依赖细胞株TF1测活和双抗夹心法ELISA测定表明:人GM-CSF基因在人肿瘤细胞中获得稳定高效表达。为进一步建立GM-CSF的转基因治疗模型提供了基础。     

人胃癌细胞系中FHIT基因mRNA的表达及pcDNA3．1-FHIT的构建与转染

目的:检测人胃癌细胞系中FHIT基因mRNA的表达状况及构建pcDNA3.1-FI-IIT真核表达载体.方法:RT-PCR法检测三种不同类型人胃癌细胞系中FHIT基因mRNA的表达,构建真核表达质粒pcDNA3.1-FHIT,通过酶切法、PCR扩增法和DNA序列分析鉴定重组质粒后,用脂质体转染至FHIT基因mR.NA阴性表达人胃癌细胞系MKN-45,经G418筛选后RT-PCR鉴定.结果:FHIT基因在人胃癌细胞系BGC-823中呈阳性表达,在MGC-803、MKN-45细胞系中呈阴性表达.FHIT基因cDNA正确克隆到真核细胞表达栽体pcDNA3.1中,并成功转染FHIT基因mRNA阴性表达人胃癌细胞系MKN-45.结论:FHIT基因在不同类型人胃癌细胞系中表达各异.成功构建pcDNA3.1-FHIT,并转染到FHIT基因mRNA阴性表达人胃癌细胞系MKN-45,使其唧T基因阳性表达.     

以可转化人工染色体(TAC)载体为基础的百脉根基因组文库的构建及筛选

可转化人工染色体(transformation-competentartificial chromosome,TAC)载体是具有克隆和转移大片段DNA特征的新型载体,是植物基因克隆和转化的有效工具.该研究把它用于豆科植物百脉根(Lotus japonicus)基因组文库的构建.此文库由1.8×105个克隆组成,平均插入片段大小为15kb左右,约覆盖百脉根基因组6倍.文库保存在12块96孔板中,每个孔中约含150个不同的重组克隆.用与花发育相关的同源基因Ljcen1片段为探针,筛选得到6个阳性克隆,酶切后验证这些阳性克隆,结果表明这些克隆含有同一个基因片段.此基因组文库可直接用于植物转化,为百脉根功能基因组的研究打下基础.     

Genomic clone of hst with transforming activity from a patient with acute leukemia

We have previously reported the identification of a novel transforming gene, hst, in DNA samples taken from human stomach cancers and a noncancerous portion of stomach. Five clones, containing the genomic hst gene, were isolated from a human cosmid library constructed from leukocyte DNA from a patient with acute leukemia. All clones possessed transforming activity when transfected to NIH3T3 cells. From one clone, an 8.7 kb BamHI fragment was subcloned into pBR322, and this subclone was active in transforming NIH3T3 cells. This is the first isolation of transformation-competent genomic hst clones directly from a human genomic library, that is, without prior passage through NIH3T3 cells.     

Activation of N-ras in a human melanoma cell line.

DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.     

A simple, effective method for the construction of subtracted cDNA libraries

A simple method is described for the construction of subtracted cDNA libraries. The technique was used to create a human pancreatic tumor cDNA library that was screened using either hybridization with cDNA probes or antibodies. cDNA from a well-differentiated tumor cell line (CD-11) was subtracted against RNA from an undifferentiated tumor cell line (Panc-1). The subtracted cDNA was purified from RNA-cDNA hybrids by oligo-dA cellulose affinity chromatography. Single-stranded subtracted cDNA was used as a template for random primed second-strand synthesis using the Klenow's fragment of DNA polymerase. After ligation with Eco R1 adapters, cDNA was inserted into lambda gt11. A library of 140,000 primary pfu was obtained that contained 92% recombinants. A small portion of this library (40,000 pfu) was subjected to probe screening with a mucin cDNA probe known to be differentially expressed by CD-11 cells. The ratio of mucin cDNA clones to actin cDNA clones was increased by greater than 300-fold in the subtracted cDNA library compared to a standard cDNA library from the same cell line. The absolute number of mucin cDNA clones per 40,000 pfu was also increased 32-fold in the subtracted library. Pancreatic tumor mucin cDNAs were also identified in the subtracted library by antibody screening. The subtraction procedure yielded a 50-fold enrichment in differentially expressed cDNA detected by antibodies, compared to a nonsubtracted library from the same cell line.     

Construction and utility of a human chromosome 22-specific Fosmid library

We have previously demonstrated the capability of the Fosmid vector based on Escherichia coli F-factor replicon to stably propagate cosmid-sized human genomic DNA fragments. Using the Fosmid vector, we have constructed and arrayed a 10 × human chromosome 22-specific library, partly by picking human positive clones from a total Fosmid library constructed using DNA from human-hamster hybrid cell line containing human chromosome 22, and partly by using flow-sorted chromosomal DNA. The clones and physical contig maps of the clones in the library will serve as a valuable resource for detailed analysis of the chromosome by providing reliable materials for high resolution mapping and sequencing. In order to efficiently built physical maps for the chromosomal regions of interest spanning several hundred kilobases to a megabase, it is necessary to rapidly identify subsets of the Fosmid clones from the library that cover such regions. In this report, we describe a method of using random amplification products derived from YAC clones to rapidly identify a subset of Fosmid clones that cover a specific genomic subregion.     

cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase

A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes. This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method. Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively. The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes. ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes. The probe also detected a sequence on chromosome 22. Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.     

Assignment of the functional gene for human adrenodoxin to chromosome 11q13----qter and of adrenodoxin pseudogenes to chromosome 20cen----q13.1.

Adrenodoxin is a small iron/sulfur protein serving as an electron-transport intermediate for all mitochondrial forms of cytochrome P450. Southern blots of normal genomic DNA cleaved with six restriction endonucleases probed with full-length human adrenodoxin cDNA revealed complex patterns indicating the presence of multiple adrenodoxin genes. Southern blots of DNA from a panel of mouse/human somatic cell hybrids identified cross-hybridizing adrenodoxin DNA in two loci, chromosome 11q13----qter and chromosome 20cen----q13.1. Examination of adrenodoxin clones from a genomic DNA library in phage lambda revealed some clones bearing gene fragments interrupted by introns and other clones bearing processed pseudogenes. By probing the mouse/human hybrids with unique intronic DNA and by correlating restriction maps of the phage clones with that of uncloned genomic DNA, we show that the authentic transcribed adrenodoxin gene lies on chromosome 11, while pseudogenes lie on chromosome 20.     

Isolation and structural mapping of a human c-src gene homologous to the transforming gene (v-src) of Rous sarcoma virus.

We have utilized a lambda Charon 4A human genomic library to isolate recombinant clones harboring a highly conserved c-src locus containing nucleotide sequences homologous to the transforming gene of Rous sarcoma virus (v-src). Four overlapping clones spanning 24 kilobases of cellular DNA were analyzed by restriction endonuclease mapping. Human c-src sequences homologous to the entire v-src region are present in a 20-kilobase region that contains 11 exons as determined by restriction mapping studies utilizing hybridization to labeled DNA probes representing various subregions of the v-src gene and by preliminary DNA sequencing analyses. A considerable degree of similarity exists between the organization of the human c-src gene and that of the corresponding chicken c-src gene with respect to exon size and number. However, the human c-src locus is larger than the corresponding chicken c-src locus, because many human c-src introns are larger than those of chicken c-src. alu family repetitive sequences are present within several human c-src introns. This locus represents a highly conserved human c-src locus that is detectable in human cellular DNAs from various sources including placenta, HeLa cells, and WI-38 cells.     

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma.

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.     

Molecular structure of the human argininosuccinate synthetase gene: occurrence of alternative mRNA splicing.

The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.