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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