STUDYING GENE EXPRESSION IN THE EMBRYO
Studying geneexpression
Most methods can be adapted to either tissue sections or whole mounts
Probe for protein ‘Western’ immunohistochemistry
Probe for mRNA ‘Northern’ hybridisation ‘in situ’ or in microarray
‘Reporter gene’ Lac Z: -galactosidaseGFP: green fluorescent protein
HYBRIDISATION - MAKING THE PROBE
(a) The normal process of transcription
mRNA made by RNA polymerase 2Base sequence equals coding strand except T > UHybridises with anti-sense RNA or cDNA
Promoter and enhancer regions Coding strand
Template strand
3’
5’5’ 3’
HYBRIDISATION - MAKING THE PROBE
(b) Production of anti-sense RNA
Anti-sense RNA made by viral polymerase acting on cloned DNABase sequence equals template strand except T > UHybridises with mRNA
Viral Promoter
3’
5’ 5’3’
Coding strand
Template strand
HYBRIDISATION – MAKING THE PROBE
(c) Production of cDNA
3’5’AAAA
cDNA made by reverse transcriptase of mRNABase sequence equals template strandHybridises with mRNA
mRNA
T T T T5’3’ cDNA
• 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 Anti-digoxigenin antibody coupled to(Digoxigenin coupled to UTP) 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 sectionor permeabilised whole mount
Probe for expressedmRNA using antisenseRNA or cDNA
Visualise locationby autoradiography,fluorescence or enzyme-linked antibody
5’ 3’
5’ 3’
1 2
3 4
Fix probe oligonucleotides representing portions of coding strands of known genes.
T T T T5’3’ cDNAs
AAAATotal cellular messenger
3’5’AAAA
Reverse transcriptase
cDNAs = template strands of DNAwith fluorescent label (F)
Degrade RNA
T5’3’
T T T
F
T
TT
TF
Hybridise
1 2
3 4
Fluorescent detection shows genes 2and 3 are expressed in this tissue
MICROARRAY
Using two probes allows comparison between tissues
RED Tissue 1
GREEN Tissue 2
YELLOW Both
BLACK Neither
IMMUNOHISTOCHEMISTRY
Fixed tissue sectionor permeabilised whole mount
Probe for expressedprotein using primaryantibody
Visualise locationusing second antibody,coupled to enzyme,fluorophore or gold
USE OF A REPORTER GENE
Engineer construct composed of regulatorysequence of interest and green fluorescent protein (GFP)gene
Inject into zygoteStudy expression ofGFP at different stagesof development
-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 ‘Western’ = Binding of antibodies to proteins
mRNA
F1 2 F
Fluorescence
mRNA
R
Ag+ AgAu
Autoradiography/Immune gold
mRNA
ES
PD
SE
PEnzyme coupled to antibody
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 Pax6- regulated genes such as lens crystallin and those genes specifying and 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
Pax6 mRNA detected by hybridisation with radioactive antisense cRNA (mouse)(Gilbert Fig. 4.17, from Grindley et al. 1995)
NF
HB100 m
Normal
FB
HB
OS 250 m
HB –HindbrainFB – Fore BrainNF – Neural foldOS – Optic stalk
8.5d
9.25d
250 m 250 m
250 m
LP
CN
RE
OV
OS
L
LP – Lens plateOV – Optic vesicleOS – Optic stalkCN – CorneaL – LensRE – Retinal epithelium
9.5d 10.5d
15.5d
IN-SITU HYBRIDISATION – WHOLE MOUNT
Mouse embryo, 10.5d
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)
Chick embryo (35h)
REPORTING pax6 REGULATORY SEQUENCES USING lacZ
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)
A B C D Pax6
Upstream enhancers of the mouse Pax6 gene
A PancreasB Lens and corneaC Neural tubeD Retina
DETECTING Pax6 PROTEIN EXPRESSION
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)
200 m
Forebrain
STUDYING THE FUNCTION OF GENES IN THE EMBRYO
Studying genefunction
Antisense RNA Blocks initiation of translation
Gene knock-out Can be constitutive or conditional
RNA interference dsRNA homologues of mRNA promote degradation of messenger
Notice that just showing where and when a gene is expressed does not give a definitive answer to its function
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
A Normal
B BMP7 knock-out(homozygote) BMP7 knock-out
(homozygote)Normal
Mouse embryos at day 17 of 21 day gestation
Conclusion:BMP7 is involved in eye development and in kidney development(Gilbert Fig. 4.20)
KNOCK-OUT OF THE BMP7 GENE
CONDITIONAL GENE KNOCK-OUT
Constitutive knock-out can be problematic in studying later effects of a knock-out if its early effects are lethal
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
Generate homozygote embryos
Induce excision enzyme at chosen stage of development to generate conditional knock-out
Inducer
Excision Enzyme
gene
Targetgene
Anti-sense RNA blocks initiation of translation of the sense messenger
Viral Promoter
3’
5’ 5’3’
Coding strand
Template strand
PROTEIN KNOCK-OUTPREVENTION OF TRANSLATION OF MESSENGER
PROTEIN KNOCK-OUT - DESTRUCTION OF MESSENGER
Antisense RNA fragment promotes cleavage of the cellular messenger
dsRNA for protein of interest is injected into a cell and is cleaved into small fragments
Antisense component of fragment associates with complementary sense sequence of mRNA using a protein called RISC
RISC
4 day mouse blastulas
Red fluorescent antibody binds to E-cadherin
4 day mouse blastulas(zygotes were injected with dsRNA for E-cadherin). This time red fluorescent antibody shows almost no reaction
PROTEIN KNOCK-OUT USE OF RNAi
Very little antibody reaction in BBlastomeres in B have failed to undergo compaction (bumpy appearance)
(Gilbert Fig. 4.23)
A
B