Gene Transfer and Expression: A Laboratory Manual

by Michael Kriegler

Overview

A practical manual of prools for achieving expression of foreign genes in mammalian cells. It includes some very new techniques such as PCR-based expression. The author gives a theoretical introduction to the prools and compares the strengths and weaknesses.

Product Details

ISBN-13:	9781349118939
Publisher:	Palgrave Macmillan UK
Publication date:	01/01/1990
Edition description:	1st ed. 1990
Pages:	242
Product dimensions:	8.27(w) x 10.98(h) x (d)

About the Author

Cytel Corporation

Table of Contents

Part 1 Overview: general comparison of vectors and DNA transfer procedures; transient and stable expression systems; viral compared to DNA mediated transfer; stratefic overview - matching experimental procedures with goals and probability of success. Part 2 Vectors (DNA): vector components - organization of the most efficient vectors such as pMT2, pCD, SRa, EBV based, BPV based and T7 based vectors; control elements; retroviral vectors and recombinant retroviruses; PCR based expression; homologous recombination and gene replacement. Part 3 Cell lines and maintenance: general tissue culture techniques; growth conditions of most commonly used mammalian cells - COS, 3t3, CV1, CHO, C127, BHK, mouse L cells, Att20, MDCK, lymphoid cells; embryonal stem cells. Part 4 DNA transfer: calcium phosphate; DEAE dextran; protoplast fusion; electroporation; chromosome mediated transfer - DEPC/PEF fusion, gamma irradiation/PEG fusion; selecting transfer technique for cell type and stability of expression. Part 5 Selection and amplification methods: DHER; Ada; Neo; GPT; Hygro; ODC, Asn synthtase, mdr; negative selection prools (gancyclovir). Part 6 Expression cloning: library construction; transfaction techniques; SIB selection. Part 7 Analysis of nucleic acid: isolation of RNA and DNA; analysis of RNA (traditional and PCR based); analysis of DNA (traditional and PCR based). Part 8 Analysis of protein: radiolabeling, immunoprecipitation and PAGE systems; western immunoblotting; analysis of glycosylation, sulfation and acylation; study of protein processing and secretion. Appendix: reagents - where to obtain and how to prepare; troubleshooting.

Editorial Reviews

"Set to become a real bench manual that should not remain gathering dust on the bookshelves of all its enthusiastic buyers. . . . many will find in this book a great deal of important information that will increase their confidence and precision during experimentation. First, the aim of the manuals is limited to specific, important techniques. Second, the book is well written: the techniques are clearly presented and up-to-date. . . . well explained and should be clear to the novice. . . . will certainly be useful in many laboratories. The organization of the methods is very logical." —Cell

"Excellent. It is written in a wonderfully direct and clear style. . . . very cost-effective. It deals with an expert area that has not been covered in detail before. The protocols themselves, the heart of any manual, are also clear and direct. . . . the author really knows they will work. . . . very comprehensive and covers the whole area of eukaryotic expression from retroviral vectors to expression cloning." —Nature

From the Publisher