Transcript L12 Differentiation and gene expression

STUDYING GENE EXPRESSION IN THE EMBRYO
Probe for mRNA
Studying gene
expression
Probe for protein
‘Reporter gene’
‘Northern’ hybridisation ‘in
situ’ or in microarray
‘Western’
immunohistochemistry
Lac Z: b-galactosidase
GFP: green fluorescent protein
Most methods can be adapted to either tissue sections or whole mounts
HYBRIDISATION - MAKING THE PROBE
(a) The normal process of transcription
mRNA made by RNA polymerase 2
Base sequence equals coding strand except T > U
Hybridises with anti-sense RNA or cDNA
Promoter and
enhancer regions
Coding strand
5’
3’
5’
3’
Template strand
HYBRIDISATION - MAKING THE PROBE
(b) Production of anti-sense RNA
Anti-sense RNA made by viral polymerase acting on cloned DNA
Base sequence equals template strand except T > U
Hybridises with mRNA
Viral Promoter
Coding strand
5’
3’
3’
Template strand
5’
HYBRIDISATION – MAKING THE PROBE
(c) Production of cDNA
cDNA made by reverse transcriptase of mRNA
Base sequence equals template strand
Hybridises with mRNA
5’
mRNA
3’
AAAA
TTTT
3’
cDNA
5’
• mRNA molecules have poly A tail
• reverse transcriptase can elongate an oligo dT primer
HYBRIDISATION – VISUALISING THE LOCATION
NATURE OF PROBE
METHOD OF DETECTION
Radiolabelled
Autoradiography
Fluorescently labelled
Fluorescence microscopy
Digoxigenin labelled
(Digoxigenin coupled to UTP)
Anti-digoxigenin antibody coupled to
alkaline phosphatase
• Can be done on fixed tissue sections or whole mounts
• Except radioactivity which is not suitable for whole mounts
• Whole mounts require permeabilisation to allow reagent access
• Digoxigenin is a plant steroid which is antigenic allowing raising of antibodies
IN-SITU HYBRIDISATION – SUMMARY
Fixed tissue section
or permeabilised
whole mount
Probe for expressed
mRNA using antisense
RNA or cDNA
Visualise location
by autoradiography,
fluorescence or
enzyme-linked antibody
MICROARRAY
Total cellular messenger
5’
3’
AAAA
Reverse
transcriptase
5’
F
3’
AAAA
TTTT
3’
5’
cDNAs
Degrade RNA
TTTT
5’
F
cDNAs = template strands of DNA
with fluorescent label (F)
3’
1
3
Hybridise
GREEN Tissue 2
5’
3’
3
4
Using two probes allows
comparison between tissues
RED Tissue 1
Fix probe oligonucleotides
representing portions of coding
strands of known genes.
2
T
Fluorescent detection shows genes 2
and 3 are expressed in this tissue
2
4
1
T
T
T
YELLOW Both
BLACK Neither
IMMUNOHISTOCHEMISTRY
Fixed tissue section
or permeabilised
whole mount
Probe for expressed
protein using primary
antibody
Visualise location
using second antibody,
coupled to enzyme,
fluorophore or gold
USE OF A REPORTER GENE
Engineer construct
composed of regulatory
sequence of interest
and green fluorescent
protein (GFP)gene
Inject into zygote
Study expression of
GFP at different stages
of development
b-galactosidase LacZ (E.coli) can also be used. An artificial substrate when
cleaved by LacZ gives a coloured insoluble product
SUMMARY OF VISUALISATION METHODS
‘Northern’ = hybridisation to mRNA
l1
l2
F
F
Fluorescence
mRNA
Ag+
‘Western’ = Binding of antibodies to proteins
Ag
Au
R
Autoradiography/
Immune gold
mRNA
E
D
mRNA
S
S
P
E
Enzyme coupled
to antibody
P
FOLLOWING GENE EXPRESSION – Pax6 AS AN EXAMPLE
What is pax6?
Pax6 encodes a transcription factor required for normal eye, nervous
and pancreatic development. It binds to enhancer elements of Pax6regulated genes such as lens crystallin and those genes specifying a
and b cells in the pancreas
Mutants in Pax6 cause severe
abnormalities
(Gilbert Fig 6.2A shows section
through developing mouse
brain in the region of the optic
cup; from Fujiwara et al., 1994)
Normal
Pax6 mutant
IN-SITU HYBRIDISATION – TISSUE SECTIONS
9.5d
8.5d
10.5d
LP
NF
OV
250 mm
HB
100 mm
250 mm
15.5d
CN
9.25d
L
FB
RE
OS
250 mm
LP – Lens plate
OV – Optic vesicle
OS – Optic stalk
CN – Cornea
L – Lens
RE – Retinal epithelium
Normal
HB
OS 250 mm
HB –Hindbrain
FB – Fore Brain
NF – Neural fold
OS – Optic stalk
Pax6 mRNA detected by hybridisation with
radioactive antisense cRNA (mouse)
(Gilbert Fig. 4.17, from Grindley et al. 1995)
IN-SITU HYBRIDISATION – WHOLE MOUNT
Mouse embryo, 10.5d
Chick embryo
(35h)
Pax6 mRNA detected by hybridisation with
digoxigenin labelled antisense RNA followed by
alkaline phosphatase-coupled antibody against
digoxigenin
(Gilbert Fig. 4.16, A from Li et al 1994, B from
Gray et al 2004)
REPORTING pax6 REGULATORY SEQUENCES USING lacZ
A
B
C
D
Pax6
Upstream enhancers of the mouse Pax6 gene
A Pancreas
B Lens and cornea
C Neural tube
D Retina
Fusion of the lens and cornea
regulatory sequence of pax6 with
the lacZ gene (mouse, 10.5d)
(Gilbert Fig 5.7, from Williams et al
1998)
DETECTING Pax6 PROTEIN EXPRESSION
Forebrain
200 mm
Mouse (9.25d)
Primary antibody:
Rabbit polyclonal antiserum raised against
Pax6
Secondary antibody:
Goat anti-rabbit antiserum with antibodies
attached to horse radish peroxidase which
can produce a coloured product
(from Mastick et al 1997)
STUDYING THE FUNCTION OF GENES IN THE EMBRYO
Notice that just showing where and when a gene is expressed
does not give a definitive answer to its function
Studying gene
function
Gene knock-out
Can be constitutive or
Antisense RNA
Blocks initiation of translation
RNA interference
dsRNA homologues of mRNA
promote degradation of
conditional
messenger
GENE KNOCK-OUT
Instead of adding genes to embryonic stem cells (as with reporter genes),
you can also replace a gene with a non-functional component.
Cloned gene cut at restriction sites and gene replaced by, for example,
an antibiotic resistance gene to aid selection of modified clone
Insertion into embryonic stem cells and selection
of heterozygotes
Injection of modified stem cells into blastula inner cell mass
Chimaeric embryos and offspring produced, some
of which have modification in germ line
Breed chimaera with wild type to produce heterozygotes
Breed heterozygotes to produce homozygotes for the knock-out
KNOCK-OUT OF THE BMP7 GENE
A
Normal
B
BMP7 knock-out
(homozygote)
Normal
BMP7 knock-out
(homozygote)
Mouse embryos at day 17 of 21 day gestation
Conclusion:
BMP7 is involved in eye development
and in kidney development
(Gilbert Fig. 4.20)
CONDITIONAL GENE KNOCK-OUT
Constitutive knock-out can be problematic in studying later
effects of a knock-out if its early effects are lethal
Inducer
Instead of replacing normal gene with non-functional
component, replace with normal gene flanked by
special recognition sequences for an inducible
excision enzyme called CRE
Target
gene
Generate homozygote embryos
Induce excision enzyme at chosen stage of development
to generate conditional knock-out
Excision
Enzyme
gene
PROTEIN KNOCK-OUT
PREVENTION OF TRANSLATION OF MESSENGER
Anti-sense RNA blocks initiation of translation of the sense messenger
Viral Promoter
Coding strand
5’
3’
3’
Template strand
5’
PROTEIN KNOCK-OUT - DESTRUCTION OF MESSENGER
dsRNA for protein of
interest is injected
into a cell and is
cleaved into small
fragments
RISC
Antisense component
of fragment associates
with complementary
sense sequence of
mRNA using a protein
called RISC
Antisense RNA
fragment promotes
cleavage of the
cellular messenger
A
4 day mouse blastulas
Red fluorescent antibody
binds to E-cadherin
PROTEIN KNOCK-OUT
USE OF RNAi
Very little antibody reaction in B
Blastomeres in B have failed to
undergo compaction (bumpy
appearance)
(Gilbert Fig. 4.23)
B
4 day mouse blastulas
(zygotes were injected with
dsRNA for E-cadherin).
This time red fluorescent antibody
shows almost no reaction