Library Science

Murray’s new handbook on Gene Transfer and Expression Protocols sets forth both current and new methodologies in a clear, concise, easy-to-follow manner, following the successful formula of the classic volumes in Humana’s Methods in Molecular Biology series. Each chapter is devoted to a thorough exposition of a single technique. An Introduction explains the significance of the protocol and provides background information. A Materials section lists all the requirements for the technique discussed. A Methods section details the procedure in a step-by-step protocol. A Notes section alerts the reader to pitfalls that may be encountered, as well as alternatives that may be used for successful completion of the experiment. Each technique is designed to guarantee optimum results. This volume is an outstanding new benchtop manual that provides protocols for introducing an isolated gene into a cell line, using various transfection techniques. Topics and techniques include: viral vectors – reporter genes – analysis of steady-state level of transcription – assay for newly initiated transcriptional complexes – immunocytological techniques – assay of structure and replication state of transfected genes – overall strategy for defining regulatory sequences Providing the latest techniques in a fast-paced area, Gene Transfer and Expression Protocols is an absolutely essential handbook for everyone involved in cytogenetics, cell genetics, molecular biology, and related fields.

Authors: Murray, Edward J.

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Table of Contents

1. Preparation of Recombinant Plasmid DNA for DNA-Mediated Gene Transfer (pp. 3-14)
   Aubin, Remy; Weinfeld, Michael; Paterson, Malcolm C.
2. Calcium Phosphate Mediated Gene Transfer into Established Cell Lines (pp. 15-22)
   Okayama, Hiroto; Chen, Claudia
3. Transfection of the Chloramphenicol-Acetyltransferase Gene into Eukaryotic Cells Using Diethyl-Aminoethyl (DEAE)-Dextran (pp. 23-34)
   Lake, Richard A.; Owen, Michael J.
4. Polybrene/DMSO-Assisted Gene Transfer (pp. 35-44)
   Aubin, Remy; Weinfeld, Michael; Paterson, Malcolm C.
5. Electroporation Technique of DNA Transfection (pp. 45-52)
   Spencer, Sarah C.
6. Irradiation and Fusion Gene Transfer (pp. 53-62)
   Goodfellow, Peter N.
7. Direct Use of lambda Phage Particles for DNA Transfection (pp. 63-80)
   Ishiura, Masahiro
8. Cationic Liposome-Mediated Transfection with Lipofectin??? Reagent (pp. 81-90)
   Felgner, Philip L.
9. Preparation of High-Molecular-Weight DNA for Use in DNA Transfection: Secondary Transfections for Cloning Active Genes by Direct Phenotypic Selection (pp. 91-98)
   Tainsky, Michael A.
10. Chromosome-Mediated Gene Transfer (pp. 99-106)
    Shipley, Janet M.; Sheer, Denise
11. Manipulation of Adenovirus Vectors (pp. 109-128)
    Graham, Frank L.; Prevec, Ludvik
12. Manipulation of Vaccinia Virus Vectors (pp. 129-146)
    Mackett, Michael
13. Manipulation of Baculovirus Vectors (pp. 147-168)
    Bailey, Mark J.; Possee, Robert D.
14. Manipulation of SV40 Vectors (pp. 169-180)
    Hammarskjold, Marie-Louise
15. Choice and Manipulation of Retroviral Vectors (pp. 181-206)
    Morgenstern, Jay P.; Land, Hartmut
16. Use of Tissue-Plasminogen Activator as a Reporter Gene (pp. 209-216)
    Wrighton, Nicholas C.
17. Use of E. coli lacZ (?÷-Galactosidase) as a Reporter Gene (pp. 217-236)
    MacGregor, Grant R.; Nolan, Garry P.; Fiering, Steven; Roederer, Mario; Herzenberg, Leonard A.
18. Application of the Firefly Luciferase Reporter Gene (pp. 237-242)
    Giguere, Vincent
19. Use of Vectors to Confer Resistance to Antibiotics G418 and Hygromycin in Stably Transfected Cell Lines (pp. 245-256)
    Santerre, Robert F.; Walls, Jenna D.; Grinnell, Brian W.
20. Selection of Cells Defective in Pyrimidine (TK???) and Purine (APRT??? and HPRT???) Salvage: Development of Host Strains Appropriate for Transfection (pp. 257-268)
    Meuth, Mark; Harwood, Janet
21. Quantitative and Qualitative Analysis of Exogenous Gene Expression by the S1 Nuclease Protection Assay (pp. 271-282)
    Walmsley, Maggie E.; Patient, Roger K.
22. The RNase Protection Assay (pp. 283-296)
    Finer, Mitchell H.
23. Primer Extension Analysis of mRNA Isolated from Transfected Cell Lines (pp. 297-306)
    Leonard, Mark W.; Patient, Roger K.
24. Northern Blot Analysis of Gene Expression (pp. 307-324)
    Krumlauf, Robb
25. Analysis of Transcriptional Initiation in Isolated Nuclei (pp. 327-336)
    Stott, David
26. Immunoperoxidase Staining of Gene Products in Cultured Cells Using Monoclonal Antibodies (pp. 339-360)
    Morris, Roger
27. The Use of Flow Cytometry to Detect Transfected Gene Products (pp. 361-378)
    Bujdoso, Raymond; Sargan, David; Ballingall, Keith; Sanderson, Andrew
28. Determination of Foreign Gene Copy Number in Stably Transfected Cell Lines by Southern Transfer Analysis (pp. 381-396)
    Fourney, Ronald M.; Aubin, Remy; Dietrich, Kevin D.; Paterson, Malcolm C.
29. Evaluation of Extrachromosomal Gene Copy Number of Transiently Transfected Cell Lines (pp. 397-404)
    Wilson, Angus C.; Patient, Roger K.
30. Use of Dpn I Restriction Enzyme to Assess Newly Replicated Gene Copies in Amplifiable Vector Systems (pp. 405-410)
    Brewer, Alison C.; Patient, Roger K.
31. Use of Polymerase Chain Reaction (PCR) to Detect Homologous Recombination in Transfected Cell Lines (pp. 411-418)
    Zimmer, Andreas; Gruss, Peter
32. Induction of Erythroid-Specific Expression in Murine Erythroleukemia (MEL) Cell Lines (pp. 421-434)
    Antoniou, Michael

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