Visualization of Gene Expression Patterns by in situ Hybridization

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 usedto 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 knownDNA sequence (compare to immunological methods)

Target sequence is detected in tissue directly i. e. in situ (compare Northern blot wherethis is not the case)

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5‘ 3‘5‘3‘

senseantisense

transcription, RNA processing

mRNA (sense)

Tissue

Gene X cDNA 5‘5‘3‘3‘

in vitro transcription

sense cRNA antisense cRNA

hybridization

Principle of in situ hybridizationU. Albrecht MM

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Fluorescent in situ hybridization (FISH) to locate DNA sequences on chromosomes

The human PER1 gene located on the short arm of chromosome 17 (17p12)

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Radioactive in situ hybridization to detect mRNA in tissues

Per3 RNA detected in a cross sectionthrough the spinal cord of a 13 day old mouse embryo.

Dorsal root ganglia (DRG)

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Colorimetric in situ hybridization to detect viral DNA/RNA

Nasopharyngeal carcinoma: in situ hybridization for Epstein-Barr virus (EBV) encoded RNAInfected cells are stained black.

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Many different forms of ISH exist and the choice of method depends on which ofthe 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

U. Albrecht MM

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.

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

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ISH provides :

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Spatial Resolution (Sections)

Expression of Ube3a in the Head of a 15.5 day mouse fetus

Spatial Resolution (Whole mount)U. Albrecht MM

Expression of Per2 in a12.5 day embryo

ZT6 ZT12 ZT18 ZT24

mPer1

mPer2

mPer3

Temporal ResolutionU. Albrecht MM

Per gene expression in the suprachiasmatic nucleus of adult mice

E6.5 E7.5

wild type

Wnt3 -/-

Genes as Tissue Markers

Brachyury is a marker forthe primitive streak andaxial mesoderm

Wnt3 is required for vertebrate axis formation

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Activity of specific alleles

m+/p- m-/p+

m = maternal allele of Ube3ap = paternal allele of Ube3a

only m-/p+ developAngelman Syndrome