Visualising Gene Expression
Reasons for studying gene expression
Where? When? How much?
a) Are 2 structures a) Is gene X expressed before a) Is gene X expression
homologous? gene Y (could X control Y)? level modulated
b) Does gene X pattern b) In a developing embryo when spatially?
structure Y? is gene X expressed – what b) Is gene X expression
c) Are genes X and Y processes could it control? level modulated
co-expressed (could c) How does expression of temporally?
they interact)? gene X change over time c) Does expression level
d) Identifying cell types (does it have multiple correspond to
functions)? phenotype?
- Exploit elements of central dogma to study gene expression
How do cells that express a gene differ from cells that don’t?
How do we detect these differences biochemically?
Detecting phenotype (using mutants) – not a generally applicable method
- Mutate Ubx gene in beetles – wild type has no legs on abdomen, mutant has legs
Ubx represses the development of legs – so normal expression domain of Ubx is
in the abdomen
- In Drosophila – mutate Ubx – 2nd thoracic segment produces wings rather than
halters – Ubx must be normally expressed in 3rd thoracic segment (normal wings)
Detecting proteins (using antibodies)
- Using the normal antibody producing mechanism in the
immune system of vertebrates, antibodies for virtually any
purified protein can be produced
- Monoclonal antibodies: Hybridoma cells produces one
specific type of antibody – select against all hybridomas –
isolate the colonies that are producing the antibody
needed
Basic Protocol using antibodies to visualise
1. Fix and permeabilise cells using a fixative (eg.
formaldehyde)
Cross-link proteins so the cell does not disintegrate
2. Incubate cells/tissue/embryo in antibody
Antibody will bind to protein of interest (ie. where
the protein is expressed)
3. Wash off excess primary antibody and incubate with
a labelled secondary antibody
Wash off unbound secondary antibody
4. Visualise labelled secondary antibody
Reasons for studying gene expression
Where? When? How much?
a) Are 2 structures a) Is gene X expressed before a) Is gene X expression
homologous? gene Y (could X control Y)? level modulated
b) Does gene X pattern b) In a developing embryo when spatially?
structure Y? is gene X expressed – what b) Is gene X expression
c) Are genes X and Y processes could it control? level modulated
co-expressed (could c) How does expression of temporally?
they interact)? gene X change over time c) Does expression level
d) Identifying cell types (does it have multiple correspond to
functions)? phenotype?
- Exploit elements of central dogma to study gene expression
How do cells that express a gene differ from cells that don’t?
How do we detect these differences biochemically?
Detecting phenotype (using mutants) – not a generally applicable method
- Mutate Ubx gene in beetles – wild type has no legs on abdomen, mutant has legs
Ubx represses the development of legs – so normal expression domain of Ubx is
in the abdomen
- In Drosophila – mutate Ubx – 2nd thoracic segment produces wings rather than
halters – Ubx must be normally expressed in 3rd thoracic segment (normal wings)
Detecting proteins (using antibodies)
- Using the normal antibody producing mechanism in the
immune system of vertebrates, antibodies for virtually any
purified protein can be produced
- Monoclonal antibodies: Hybridoma cells produces one
specific type of antibody – select against all hybridomas –
isolate the colonies that are producing the antibody
needed
Basic Protocol using antibodies to visualise
1. Fix and permeabilise cells using a fixative (eg.
formaldehyde)
Cross-link proteins so the cell does not disintegrate
2. Incubate cells/tissue/embryo in antibody
Antibody will bind to protein of interest (ie. where
the protein is expressed)
3. Wash off excess primary antibody and incubate with
a labelled secondary antibody
Wash off unbound secondary antibody
4. Visualise labelled secondary antibody
