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Genes & Development Part 2: Molecular Methodology to Investigate Differential Gene Expression RNA Analysis – Northern Blots • Isolate total or pA+ RNA • Separate by size with electrophoresis (special gel to prevent secondary structure) • Transfer electrophoresed RNA to nitrocellulose/nylon membrane (blot) • Probe blot with labeled cDNA probe • Northern analysis requires that portion of gene sequence be known (gene be cloned) Northern Analysis: Gel Electrophoresis Formaldehyde gels Methyl-mercury-OH gels Northern Analysis: ElectroBlotting Anode (-) Support pad RNA gel Nylon or nitrocellulose filter Cathode (+) Support pad Northern Analysis: ElectroBlotting 100V Northern Analysis: Probing RNA Localization • Developmental Northerns – Temporal expression information – Spatial expression information (limited to microdissected embryo regions and tissues) – Requires RNA species be fairly abundant RNA Localization Developmental Stages RNA Localization Developmental Stages RNA Localization in Tissues RNA Expression Following Experimental Treatments RNA Expression Following Experimental Treatments RNA Analysis RT-PCR – Isolate RNA (total or pA+) – Reverse transcribe using oligo dT primer – Amplify cDNA by PCR using gene specific primers and using only 10-15 rounds – Run PCR product on agarose gel – Southern blot and probe with cDNA probe – more sensitive than northern blot RT-PCR Muscle Actin Gsc FGF: - + - + XBra XNot XBra XNot - + - + - + - + - Muscle Actin Gsc + - + RNA - Sequencing • Isolate mRNA from different stages/tissues • Convert mRNAs to cDNAs RNA Sequencing • Fragment cDNAs, and add adapters of known sequence • Amplify frags by PCR • Sequence PCR frags, map back to genome to determine which genes are expressed RNA Localization • Whole mount in situ hybridization allows us to see which cells express a given mRNA. • Hybridize antisense RNA probe to mRNA in embryo • Information: temporal and spatial expression pattern • Putative function of gene product based on expression pattern Whole-mount in situ hybridization Whole mount in situ hybridization m FGF8 expression in 3-day chick. Expression detected in somites (s), limb buds (l), brachial arches (b) & midbrain-hindbrain boundary (m) b s l l Whole Mount In Situs EphA4 EphB3 En-1 Whole Mount In Situs EphA4 Microarray Analysis Microarray Analysis Microarray Analysis of Xnr1-Induced Genes in Xenopus Animal Caps Xnr1 Induced Genes Biochemical & Immunological Techniques • Biochemical purification & characterization • Generation of antibodies that specifically recognize proteins – pAb – polyclonal antibodies • Many Abs varieties generated against a protein • Each Ab may recognize a unique epitope – mAb – moloclonal antibodies • One Ig producing cell (B-cell) reacts to protein • One variety of Ab produced which recognizes one specific epitope Biochemical Techniques • Classical protein biochemistry – Column chromatography – Preparative electrophoresis • Amino acid sequencing • Structural Biochemistry – X-ray crystallography, CD-ORD, MS-AS, NMR – Computer generated structure predictions Immunological Techniques • Immunohistochemistry (whole mount & section) • Immunolocalization • Western analysis • Immunoprecipitation IP • Co-immunoprecipitation coIP • cDNA expression library screening Western Analysis • Separate all proteins by electrophoresis (SDS-PAGE) – Transfer proteins to membrane – Incubate with antibody to specific protein and detect presence/absence • IP protein using Ab to first protein – Run gel and probe with 2nd Ab to second protein to determine if two proteins interact Western Analysis Identifying Developmentally Significant Genes • Differential Screens • Homology Screens • Functional Screens Require generation of cDNA libraries followed by screening for desired clones • Mutagenic Screens Requires an assayable phenotype and viable heterozygote (doesn’t work well for dominant lethal mutations) Differential Screening • Use of a subtracted probe to screen a tissue specific cDNA library • Subtracted probe is not gene-specific rather it is differentiation-state-specific probe • cDNA made from one tissues mRNA • cDNA is hybridized to mRNA from another related yet different cell type to remove all transcripts in common (housekeeping genes) • Probe has then has common RNA transcripts subtracted from it Differential Screening: Subtracted Library Epidermal cells Neuroblasts & Neuroectoderm pA+ RNA pA+ RNA 1st strand cDNA Anneal epi RNA + neuro cDNA (2X) recover ss cDNA from hydroxyapatite column (neuro + epi specific cDNAs) Anneal ss cDNAs from HA column to neuro mrna recover ds cDNA/mRNA hybrids from HA column (neuro specific cDNAs) Use neuro specific cDNAs to make library Differential Screen: Subtracted Probe & +/- Screen Epidermal cells Neuroblasts & Neuroectoderm pA+ RNA pA+ RNA 1st strand cDNA (32P-label) Anneal epi RNA + neuro cDNA recover ss cDNA (HA column) (neuro + epi specific cDNAs) Anneal ss cDNAs from HA column to unlabeled,neuro mRNA recover ds cDNA/mRNA hybrids from HA column (neuro specific cDNAs) Use labeled cDNA as - probe to screen library Use labeled cDNA as + probe to screen library Differential Screen: Subtracted Probe & +/- Screen Plus-Probe Minus-Probe Homology Screens • Screen a cDNA library for a related gene from a different species • Reduce stringency of hybridization to compensate for slight differences in gene sequences between the two species • Find a gene in Drosophila – antennapedia • Is there a related gene in mammals? – Hox genes Functional Screens • Add back mRNAs to rescue a mutant phenotype – Drosophila • Cloning of bicoid by adding back fractions of mRNAs from wt eggs to bicoid mutant eggs – Xenopus • Cloning of noggin by adding back mRNAs to ventralized embryos • Screening of expression libraries • Look for functional interactions – two hybrid Functional Cloning • Cloning via DNA interaction or protein interaction – Screen cDNA expression library with DNA probe to identify DNA binding proteins – Screen cDNA expression library by yeast twohybrid to identify protein-protein interactions – Screen cDNA expression library by transfection into cultured cells Functional Screen: Yeast 2Hybrid • Fuse DNA binding domain to bait – cDNA coding DNA binding domain of yeast txn factor – Ligated in-frame with cDNA coding protein of interest – bait • Make cDNA library in vector where cDNAs will be in-frame with txn activation domain of yeast txn factor – This is the prey • If bait and prey proteins interact, txn-activation & DNA-binding domains of yeast txn factor will be next to each other and will drive expression of gene in yeast that allows yeast to live Functional Screen: Yeast 2Hybrid Prey UnkP UnkP Bait UnkP LexA-DBD POI UnkP Transformed into yeast Gal4-AD UnkP Gal4-AD Gal4-AD Gal4-AD Gal4-AD UnkP Gal4-AD Transform yeast cells expressing bait with cDNA prey library. Each yeast cell will make 1 of the prey fusion proteins Functional Screen: Yeast 2Hybrid When bait & prey interact, functional txn factor is formed and expression of His3 occurs Prey Bait RP LexA Site His3 Mutagenic Screens: Chemical/Radiation • • • • Mutagenize one sex (usually male) (ENU; EMS; X-ray) Mate to wt female All progeny (F1) are heterozygotes for any mutations Outcross all F1 females to wt males or backcross FI males to mother – 50% of F2 will be heterozygotes • Testcross F2 sibs to produce – 25% with mutant phenotype • Outcrossed F1 becomes founder for further generations to examine and clone mutated gene Mutagenic Screens: Insertional Mutagenesis • Create transgenic animals with heterologous marker gene inserted randomly into genome – LacZ or GFP most common marker gene • Screen for animals with mutant phenotype • Identify location of insertion and clone surrounding sequences – Use probe corresponding to marker gene sequences Functional Analysis of Developmental Genes • Mutant phenotype associated with gene • Generation of mutant phenotype when not already known – Targeted disruption (transgenic analysis) – Mis-expression • Ectopic expression • Over expression – Biochemical analysis • Subcellular location • Protein-protein interactions • Enzymology Transgenic Analysis • Random insertion of transgenes (for mutagenesis) • Targeted insertion of transgenes – Knockout – Knockin • Requires special vectors – contains flanking sequences to permit homologous recombination between construct and chromosome – Contains selectable marker to permit survival only of homologous recombination and not non-homologous Transgenic Analysis Vector for homologous recombination Transgenic Analysis Gene of interest Transgenic Analysis neor Homologous recombination replaces region of gene with neomycin resistance gene and disrupts generation of functional protein. Neor allows for cells to be selected for using antibiotic neomycin. Transgenic Analysis HSV-tk neor Non-homologous recombination inserts thymidine kinase. The presence of gene allows cells containing it to be killed by the thymidine analog gancyclovir or FIAU. Only HSV (viral) tk will phosphorylate the nucleotide analog so only the cells with HSV-tk will be killed. The phosphorylated analog stops DNA synthesis when it is incorporated by DNA polymerase. Transgenic Analysis iodo arabinose fluoro Transgenic Analysis Transgenic Analysis & FIAU insensitivity Transgenic Analysis Transgenic Analysis Morphological Analysis of Bmp7 Knockout Mice Functional Analysis by Misexpression • Ectopic expression – Express protein in cells which would not normally have the protein present • Mis-expression at different stages which could be in normal or ectopic locations • Drosophila, Xenopus, zebrafish, C. elegans, cultured cells (ES cells mouse) – Systems conducive to DNA/RNA injection/transfection/infection Misexpression • Allows expression of wt or mutant forms of protein • Allows knockout using antisense RNA, antisense oligos, or dominant-negative proteins • Allows increase in activity of protein by expressing constitutively active proteins Misexpression • Injection of either synthetic mRNAs or DNA expression vectors • Use of DNA expression vector requires specific regulatory elements • Judicious use of regulatory elements allows one to define the expression pattern – Use homologous elements – expressed in wt pattern – Use heterologous elements – expression in pattern of gene from which element came – Use of constitutive heterologous elements (viral LTR or housekeeping gene – drive expression everywhere in embryo Biochemical Analysis • Function of secreted morphogens by exposing whole embryo, explants or cultured cells to purified morphogen • Determining differentially expressed genes by detecting differences in protein expression – 2D SDS-PAGE • Separate proteins based on isoelectric point then by size • Compare proteins from two cell types to identify unique proteins • Purify protein, sequence, reverse transcribe oligonucleotide, screen cDNA library Biochemical Analysis: Bioactivity Biochemical Analysis: 2D Gel IEF Gel B Acidic - Basic + SDS-PAGE A Biochemical Analysis: 2D Gel Compare 2D Gels of proteins form two cell types Cell type A Cell type B Biochemical Analysis:2D Gel Overlay 2D gels to identify cell-type-specific proteins