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Visualization of Gene Expression Patterns by in situ Hybridization U. Albrecht MM U. Albrecht MM I. Introduction 1) What is in situ hybridization 2) Why in situ hybridization U. Albrecht MM 1) What is in situ hybridization ? The principle behind in situ hybridization (ISH) is the specific annealing of a labelled nucleic acid probe to complementary sequences in fixed tissue, followed by visualization of the location of the probe. This technique can be used to locate DNA sequences on chromosomes, to detect RNA or viral DNA/RNA. x Advantages of ISH: speed with which specific probes for ISH can be generated from fragments of known DNA sequence (compare to immunological methods) Target sequence is detected in tissue directly i. e. in situ (compare Northern blot where this is not the case) U. Albrecht MM Principle of in situ hybridization Gene X cDNA 5‘ 3‘ DNA, oligos 3‘ 5‘ sense cRNA antisense cRNA hybridization Tissue mRNA (sense) transcription, RNA processing sense antisense 5‘ 3‘ 3‘ 5‘ hybridization on chromosome in vitro transcription U. Albrecht MM Fluorescent in situ hybridization (FISH) to locate DNA sequences on chromosomes The human PER1 gene located on the short arm of chromosome 17 (17p12) U. Albrecht MM Radioactive in situ hybridization to detect mRNA in tissues Dorsal root ganglia (DRG) Per3 RNA detected in a cross section through the spinal cord of a 13 day old mouse embryo. U. Albrecht MM Colorimetric in situ hybridization to detect viral DNA/RNA Nasopharyngeal carcinoma: in situ hybridization for Epstein-Barr virus (EBV) encoded RNA Infected cells are stained black. U. Albrecht MM Many different forms of ISH exist and the choice of method depends on which of the following are important for the specific application: 1. Sensitivity: - accessibility of the target RNA or DNA (ss or ds) - probe labelling and detection - length of probe - non specific background 2. Resolution: - depends on probe labelling and detection. Can vary from subcellular to greater than a cell diameter. 3. Specificity: - stringency washing - similarity between probe and sequence (conserved regions of gene families) - oxidative state of tissue, chemical history of tissue 4. Simultaneous detection: - probes labelled by different methods - combining ISH with IHC 5. Safety: for large scale analysis non-radioactive probes safer than radioactive probes 6. 3D patterns: - reconstruction by serial sections - whole mount ISH U. Albrecht MM I. Introduction 1) What is in situ hybridization 2) Why in situ hybridization U. Albrecht MM 2) Why in situ hybridization Genome research concerns the function and interaction of genes and gene products. Clues for function of a gene: - spatial and temporal activation of a specific gene in the wild type organism. Gives information on where and when the gene is important. - changes of the above patterns in genetically altered organisms reveals genetic interactions For example in several model organisms such as Drosophila melanogaster a number of complex genetic regulatory networks have been unravelled and embryonic development is now understood in terms of an orchestrated expression of developmental genes. U. Albrecht MM ISH provides : 1. Spatial resolution of gene expression 2. Temporal resolution of gene expression 3. Genetic interactions of genes, gene relationships in specific diseases, genes as tissue markers 4. Activity of specific alleles U. Albrecht MM Spatial Resolution (Sections) Expression of Ube3a in the Head of a 15.5 day mouse fetus U. Albrecht MM Spatial Resolution (Whole mount) Expression of Per2 in a 12.5 day embryo U. Albrecht MM Temporal Resolution mPer1 mPer2 mPer3 ZT6 ZT12 ZT18 Per gene expression in the suprachiasmatic nucleus of adult mice ZT24 U. Albrecht MM Genes as Tissue Markers E6.5 E7.5 Brachyury is a marker for the primitive streak and axial mesoderm wild type Wnt3 is required for vertebrate axis formation Wnt3 -/- U. Albrecht MM Activity of specific alleles m+/p- m = maternal allele of Ube3a p = paternal allele of Ube3a m-/p+ only m-/p+ develop Angelman Syndrome