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Transcript
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