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Certaines tumeurs expriment des antigènes par des CTLs isolés à partir des lymphocytes des patients. Ces antigènes, tout comme les antigènes d’histocompatibilité mineurs et les antigènes tum-de souris, n’induisent pas de réponse à anticorps. C’est entre autres le cas du mélanome du patient MZ2, pour lequel on a pu définir l’expression de six antigènes spécifiques à l’aide de clones CTL autologues. Nous avons décidé de cloner le gène qui permet l’expression d’un de ces antigènes, l’antigène E.
Comme première étape du clonage, nous avons cotransfecté l’ADN génomique d’une cellule exprimant l’antigène E avec le plasmide sélectionnable pSVtkneoβ, dans une cellule réceptrice qui n’exprimait pas l’antigène E. Parmi 700.000 transfectants résistants à la généticine, nous avons ainsi obtenu 1 transfectant qui exprimait l’antigène E. Nous avons distingué ce transfectant du reste des cellules résistantes à l’antibiotique de sélection grâce à sa capacité de stimuler la production de Tumor Necrosis Factor (TNF) par un clone CTL anti-E.
Nous avons ensuite vérifié que l’antigène exprimé provenait bien de l’ADN transfecté. Pour cela, nous avons sélectionné des variants de perte d’antigène, Nous avons observé que ces variants de perte avaient perdu également des séquences capables d’hybrider une sonde contenant le gène néor.
La perte simultanée de l’expression de l’antigène E et de séquence néor montre que les gènes étaient voisins, car une délétion portant sur l’un d’eux a également affecté l’autre. C’est ce que l’on observe dans le cas de gènes cotransfectés, qui s’intègrent probablement ensemble en un endroit du génome.
Le clonage du gène E va nous permettre d’aborder les gènes permettant l’expression d’antigènes de rejet par des tumeurs humaines. Nous espérons mieux connaître leur spécificité, leur relation possible avec le processus de transformation cancéreuse et leur potentiel éventuel pour l’immunothérapie.
T-Lymphocytes, Cytotoxic --- Gene Library --- Cloning, Molecular --- Medical Laboratory Science
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Combinatorial chemistry --- Chemistry, Pharmaceutical --- Drug Design --- Gene Library --- Pharmaceutical Preparations --- Chimie combinatoire --- Synthese (chemie) --- chemical synthesis --- JEX7 --- Chemistry, Pharmaceutical. --- Drug Design. --- Gene Library. --- chemical synthesis. --- Chemistry - Combinations thereof --- Chemistry - Combinations thereof. --- Chemistry --- Engineering --- Health Sciences --- Life Sciences --- Material Science and Metallurgy --- General and Others --- Biotechnology --- Chemical Engineering --- Pharmacy and Pharmacology --- Composites
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Chemistry, Pharmaceutical --- Drug Design --- Gene Library --- Pharmaceutical Preparations --- Combinatorial chemistry --- Pharmaceutical chemistry --- Chimie combinatoire --- Chimie pharmaceutique --- chemical synthesis --- Periodicals. --- Periodicals --- Périodiques --- Synthese (chemie) --- JEX7 --- Chemistry. --- Chemistry --- Engineering --- Health Sciences --- Life Sciences --- Material Science and Metallurgy --- General and Others --- Biotechnology --- Chemical Engineering --- Pharmacy and Pharmacology --- Composites
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Descriptive organic chemistry --- Technology, Pharmaceutical --- Combinatorial Chemistry Techniques --- Gene Library --- Solid-phase synthesis --- Combinatorial chemistry --- 541.5 --- 615.015.2 --- #WSCH:LOSA --- Organic compounds --- Combinatorial organic synthesis --- Combinatorial synthesis (Organic chemistry) --- High-throughput organic synthesis --- High-throughput synthesis (Organic chemistry) --- Multiple parallel synthesis (Organic chemistry) --- Pharmaceutical chemistry --- Biochips --- Valencies. Bonds. Affinity --- Combined effects. Action of combination of drugs --- Synthesis --- Combinatorial chemistry. --- Solid-phase synthesis. --- 615.015.2 Combined effects. Action of combination of drugs --- 541.5 Valencies. Bonds. Affinity
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Genetic engineering --- DNA, RECOMBINANT --- Génie génétique --- genetic engineering --- DNA, Recombinant. --- Genetic Engineering. --- Engineering, Genetic --- Intervention, Genetic --- Genetic Intervention --- Genetic Interventions --- Interventions, Genetic --- Biotechnology --- Cloning, Molecular --- DNA, Recombinant --- Industrial Microbiology --- Artificial Gene Fusion --- Organisms, Genetically Modified --- Animals, Genetically Modified --- Plants, Genetically Modified --- Recombinant DNA Research --- Recombination Joint --- Genes, Spliced --- Recombinant DNA --- DNA Research, Recombinant --- Joint, Recombination --- Research, Recombinant DNA --- Spliced Genes --- DNA Transposable Elements --- Genetic Engineering --- Genetic Vectors --- Genomic Library --- Gene Library --- Genetic engineering. --- Molecular cloning.
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This manual is an indispensable tool for introducing advanced undergraduates and beginning graduate students to the techniques of recombinant DNA technology, or gene cloning and expression. The techniques used in basic research and biotechnology laboratories are covered in detail. Students gain hands-on experience from start to finish in subcloning a gene into an expression vector, through purification of the recombinant protein.The second edition has been completely re-written, with new laboratory exercises and all new illustrations and text, designed for a typical 15-week semester,
Molecular biology --- Technique --- DNA, Recombinant. --- Molecular Biology --- Protein Engineering. --- methods. --- Genetic Engineering, Protein --- Proteins, Genetic Engineering --- Genetic Engineering of Proteins --- Engineering, Protein --- Engineering, Protein Genetic --- Protein Genetic Engineering --- Recombinant DNA Research --- Recombination Joint --- Genes, Spliced --- Recombinant DNA --- DNA Research, Recombinant --- Joint, Recombination --- Research, Recombinant DNA --- Spliced Genes --- Molecular biochemistry --- Molecular biophysics --- Genetic Engineering --- Directed Molecular Evolution --- DNA Shuffling --- Molecular Farming --- Cloning, Molecular --- DNA Transposable Elements --- Genetic Vectors --- Genomic Library --- Gene Library --- Artificial Gene Fusion --- Biochemistry --- Biophysics --- Biomolecules --- Systems biology
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The two volumes of Essential Molecular Biology: A Practical Approach provide clear theoretical and practical guidance to the fundamental techniques of recombinant DNA analysis. Although of particular value to research workers entering the field for the first time, the protocols described will also be of interest to experienced researchers. Volume I concentrates on the preparation, purification and handling of nucleic acids, and includes chapters on microbiological procedures and the construction and use of recombinant DNA molecules. Volume II covers the procedures needed for identification of specific genes and the study of their structure and cellular expression. Topics covered include preparation of gene libraries, hybridization methods, nucleic acid sequencing, DNA mapping, and DNA amplification by the polymerase chain reaction. Each chapter is written in an accessible style by an acknowledged expert in that field with the emphasis on providing sound technical instruction, in combination with the necessary theoretical background and useful troubleshooting guidance. This book thus brings powerful techniques at the forefront of molecular biology research to the researcher. Together, the two volumes provide a comprehensive molecular biology manual tailored specially for the novice.
Molecular cloning. --- Recombinant DNA. --- Cloning, Molecular. --- Molecular Cloning --- Cosmids --- Cloning, Organism --- DNA, Recombinant. --- 577.2 --- #WSCH:WBIO --- 577.2 Molecular bases of life. Molecular biology --- Molecular bases of life. Molecular biology --- Recombinant DNA Research --- Recombination Joint --- Genes, Spliced --- Recombinant DNA --- DNA Research, Recombinant --- Joint, Recombination --- Research, Recombinant DNA --- Spliced Genes --- Cloning, Molecular --- DNA Transposable Elements --- Genetic Engineering --- Genetic Vectors --- Genomic Library --- Gene Library --- Artificial Gene Fusion --- Molecular biology --- Biologie moléculaire --- DNA. --- DNA --- Bacteriophages --- Rna
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The first libraries of complementary DNA (cDNA) clones were con structed in the mid-to-late 1970s using RNA-dependent DNA polymerase (reverse transcriptase) to convert poly A* mRNA into double-stranded cDNA suitable for insertion into prokaryotic vectors. Since then cDNA technology has become a fundamental tool for the molecular biologist and at the same time some very significant advances have occurred in the methods for con structing and screening cDNA libraries. It is not the aim of cDNA Library Protocols to give a comprehensive review of all cDNA library-based methodologies; instead we present a series of up-to-date protocols that together should give a good grounding of proce dures associated with the construction and use of cDNA libraries. In deciding what to include, we endeavored to combine up-to-date versions of some of the most widely used protocols with some very usefiil newer techniques. cDNA Library Protocols should therefore be especially useful to the investigator who is new to the use of cDNA libraries, but should also be of value to the more experienced worker. Chapters 1—5 concentrate on cDNA library construction and manipula tion, Chapters 6 and 7 describe means of cloning difficult-to-obtain ends of cDNAs, Chapters 8-18 give various approaches to the screening of cDNA libraries, and the remaining chapters present methods of analysis of cDNA clones including details of how to analyze cDNA sequence data and how to make use of the wealth of cDNA data emerging from the human genome project.
DNA, Complementary --- Gene Library --- Genetic Techniques --- Antisense DNA --- DNA --- Gene libraries --- Molecular cloning --- ADN --- Clonage moléculaire --- Laboratory manuals --- Manuels de laboratoire --- 57.088 --- 577.212.3 --- -DNA --- -Gene libraries --- -Molecular cloning --- -#ABIB:aimm --- Cloning, Molecular --- DNA cloning --- Gene cloning --- Cloning --- Genetic engineering --- Molecular genetics --- Clone cells --- Gene banks (Genetic engineering) --- Genomic banks --- Genomic libraries --- Biological resource centers --- Genomes --- Recombinant DNA --- Deoxyribonucleic acid --- Desoxyribonucleic acid --- Thymonucleic acid --- TNA (Nucleic acid) --- Deoxyribose --- Nucleic acids --- Genes --- Complementary DNA --- Antisense nucleic acids --- Special methods and techniques for studing biological molecules. Separation. Centrifuging. X-ray study. Radioisotope methods --- Nucleic acid base and sequence compositon. Experimental deciphering of genetic code. --- Antisense DNA -- Laboratory manuals. --- DNA -- Laboratory manuals. --- Electronic books. -- local. --- Gene libraries -- Laboratory manuals. --- Molecular cloning -- Laboratory manuals. --- DNA, Single-Stranded --- DNA Probes --- Genetic Structures --- Investigative Techniques --- Nucleic Acid Probes --- Analytical, Diagnostic and Therapeutic Techniques and Equipment --- Genetic Phenomena --- Nucleic Acids --- Phenomena and Processes --- Molecular Probes --- Laboratory Chemicals --- Nucleic Acids, Nucleotides, and Nucleosides --- Chemicals and Drugs --- Specialty Uses of Chemicals --- Chemical Actions and Uses --- Genetics --- Biology --- Health & Biological Sciences --- DNA, Complementary. --- Gene Library. --- Genetic Techniques. --- Laboratory manuals. --- 577.212.3 Nucleic acid base and sequence compositon. Experimental deciphering of genetic code. --- 57.088 Special methods and techniques for studing biological molecules. Separation. Centrifuging. X-ray study. Radioisotope methods --- Antisense dna --- Dna --- Dna, complementary. --- Gene library. --- Genetic techniques. --- Clonage moléculaire --- #ABIB:aimm --- Nucleic acid base and sequence compositon. Experimental deciphering of genetic code --- Cytology. --- Immunology. --- Cell Biology. --- Immunobiology --- Life sciences --- Serology --- Cell biology --- Cellular biology --- Cells --- Cytologists
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Molecular biology --- DNA, Recombinant. --- Transduction, Genetic. --- Transfection. --- Transformation, Genetic. --- Genetic Transformation --- Genetic Transformations --- Transformations, Genetic --- Crosses, Genetic --- Transduction, Genetic --- Transfection --- Transfections --- Transformation, Bacterial --- Transformation, Genetic --- Genetic Transduction --- Genetic Transductions --- Transductions, Genetic --- Recombinant DNA Research --- Recombination Joint --- Genes, Spliced --- Recombinant DNA --- DNA Research, Recombinant --- Joint, Recombination --- Research, Recombinant DNA --- Spliced Genes --- Cloning, Molecular --- DNA Transposable Elements --- Genetic Engineering --- Genetic Vectors --- Genomic Library --- Gene Library --- Artificial Gene Fusion --- Conferences - Meetings --- Bacteriophages --- Genetic transformation --- Transduction --- DNA --- TRANSDUCTION --- TRANSFECTION --- TRANSFORMATION --- Congresses. --- Recombinant --- congresses. --- GENETIC --- Dna --- Transformation --- Genetic --- DNA, Recombinant
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