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Analysis of large libraries of protein mutants using flow cytometry
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Year: 2001

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GENE LIBRARY


Book
Transfection d'une bibliothèque génomique en vue d'isoler le gène qui permet l'expression de l'antigène E du mélanome humain MZ2-MEL

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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.


Book
Molecular biology techniques : a classroom laboratory manual
Authors: --- ---
ISBN: 128331097X 9786613310972 0123855454 0123855446 9780123855459 9780123855442 6613310972 9781283310970 Year: 2012 Publisher: Amsterdam ; Boston : Elsevier /Academic Press,

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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,


Book
Essential molecular biology : a practical approach
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ISBN: 019963114X 0199631158 0199631115 0199631107 0199631123 0199631131 Year: 1991 Publisher: Oxford : IRL Press,

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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.

cDNA library protocols
Authors: ---
ISBN: 089603383X 9786610836765 1280836768 1592595553 Year: 1997 Volume: 69 Publisher: Maastricht : University of Limburg. Institute for human psychopharmacology,

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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.

Keywords

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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