Retinoid Protocols, Softcover reprint of the original 1st ed. 1998 Methods in Molecular Biology Series, Vol. 89

Interest in retinoic acid, the main biologically active derivative of vi- min A or retinol, increased dramatically between 1989 and 1993, following the cloning of nuclear receptors or RARs reported in 1987 (Fig. 1). Important discoveries since then have shown how RARs work as all-trans retinoic ac- dependent heterodimers with related nuclear receptors for 9-cis retinoic acid called RXRs. This has stimulated the development of synthetic analogs s- cific for each type of receptor, and opens the way to develop new methods for regulating pharmacologically the activity ofretinoic acid-dependent pathways of gene activation. The potential for the development of new drugs by the pharmaceutical industry is now a maj or factor driving forward our understa- ing of vitamin A-regulated pathways in animal development and homeostasis. However, apart from the real potential ofretinoid analogs as novel pharma- logical agents, there remains the considerable intellectual challenge of und- standing the way in which vitamin A and its derivatives function in cell development and differentiation. Retinoid Protocols is an attempt to bring together various methodologies that will be vital for rising to this challenge in the future. Retinoid molecular biology has few methods of its own, but is reliant on standard molecular biology methods applied to this particular research area.

Handling and Analysis of Retinoids.- Properties of Retinoids.- Quantitative Analyses of Naturally Occurring Retinoids.- Detection and Measurement of Retinoic Acid Production by Isolated Tissues Using Retinoic Acid-Sensitive Reporter Cell Lines.- Retinoid-Binding Proteins.- Immunohistochemistry for CRBPs and CRABPs.- Whole-Mount In Situ Hybridization of Mouse Embryos Exposed to Teratogenic Levels of Retinoic Acid.- Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for Cellular Retinoid-Binding Proteins.- Methods for Producing Recombinant Human Cellular Retinaldehyde-Binding Protein.- Expression and Purification of CRABPs from E. coli.- Purification and Fluorescent Titration of Cellular Retinol-Binding Protein.- Fluorometric Titration of the CRABPs.- Expression and Mutagenesis of Retinol-Binding Protein.- Interactions of Retinol-Binding Protein with Transthyretin and Its Receptor.- Detection of Conformational Changes in Cellular Retinoid-Binding Proteins by Limited Proteolysis.- Measurement of Rates of Dissociation of Retinoids from the Interphotoreceptor Retinoid-Binding Protein.- Use of Antisense Oligonucleotides to Study the Role of CRABPs in Retinoic Acid-Induced Gene Expression.- Nuclear Retinoid Receptors.- Preparation of Polyclonal Antibodies to Retinoid Receptors.- Detection of RARs and RXRs in Cells and Tissues Using Specific Ligand-Binding Assays and Ligand-Binding lmmunoprecipitation Techniques.- Nonisotopic In Situ Hybridization for the Detection of Nuclear Retinoid Receptor Transcripts in Tissue Sections.- In Situ Hybridization with 35S-Labeled Probes for Retinoid Receptors.- Isolation of Retinoid Receptors from Manimalian Cells.- Analysis of Retinoid Receptor Phosphorylation.- Photoaffinity Labeling of RARs and Mapping of Labeled Sites by an Endoproteinase Combination Technique.- PCR Cloning of N-Terminal RAR Isoforms and APL-Associated PLZF-RAR? Fusion Proteins.- RT-PCR in Diagnosis and Disease Monitoring of Acute Promyelocytic Leukemia (APL).- A Two-Hybrid Protein Interaction System to Identify Factors That Interact with Retinoid and Vitamin D Receptors.- Gel-Shift Analysis and Identification of RXREs and RAREs by PCR-Based Selection.- Identification and Cloning of RA-Regulated Genes by mRNA-Differential Display.- Gene Targeting of Retinoid Receptors.