Gene Transfer and Expression: A Laboratory Manual

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 protocols (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.