1Lecture 15 Functional GeneticsFunctional genomics: Identify the function of each and every gene in the genome. Since the characterization of the function of a protein domain in one organism generally provides hint to its function in another organism, the first goal of functional genomics is to identify as many genes as possible in major model organismsBasic ApproachesA. Forward genetics: Random mutagenesis, screen for traits of interests Chromosome walking or transposon-taggingB. Gene expression profile (analyses of transcriptome) C. Reverse genetics: disrupt a particular gene or set of genes with known seq.D. Fine structure genetics2Forward Forward andand reversereverse geneticsgenetics• Forward genetics starts with identification ofinteresting mutants– Then aims to discover the function of genes defective inmutants• Reverse genetics starts with a known gene andalters its function– Then aims to determine the role of the gene from theeffects on the organism3Reverse genetic methods• RNA interference• Identify gene affected byInsertional or chemical mutagenesisThen screen for the mutation• Delete genes by homologous recombinationCan be done in yeast, mouse and flies4RNA interferenceRNA interferenceRNAi movie www.nature.com/focus/rnai/animations/index.htmlpost-transcriptional gene suppression (PTGS)•Initially characterized in:–C. elegans•Double-stranded RNA injection-named RNAi –Plants •Resistance to spread of virus•Suppression of transgene expression•Function of RNAi likely used to detect:–genome-invading transposable genetic elements and double-stranded (ds) RNA viruses–Other abnormal gene expression5Diverse organisms display RNAiDiverse organisms display RNAi• Model animals (Drosophila, C. elegans, mouse, etc.)• Non-model animals (cnidaria, beetles, crickets,crustaceans)• Protozoa (e.g. Tetrahymena)• Dictyostelium• Plants (e.g. Arabidopsis, maize)• Fungi (e.g. Neurospora)6Fig. C.9(RDRP)7Potential Practical Applicationsof RNA Interference• Control virus infection• Analysis of cell biology by silencingspecific gene• Target validation for drug development• Potentially new therapeutic approachesto treating diseases - a new approach toantisense and new possibilities for genetherapy889Challenges for siRNAsas a therapeutic agentChallenges for siRNAsas a therapeutic agentCellular uptake in tissue under physiological conditions Cellular uptake in tissue under physiological conditions a) Chemical modifications to improve 1) Stability in blood 2) Tissue redistribution and cellular transport 3) Efficient silencing of gene b) Specific target site delivery Examples, eye and CNS c) Delivery as a complex10Systematic RNAi screens inSystematic RNAi screens inC.C. elegans elegans and mammalian cellsand mammalian cells• In the nematode, C. elegans, RNAi is easy to do– Inject dsRNA– Feed bacteria expressing dsRNA– Or soak in solution of dsRNA• Makes systematic RNAi screens possibleFraser, 2000-Chromosome I-feedingGonczy, 2000-Chromsome III-injectionKamath, 2003-Genome-wide feedingSonnichensen, 2005-Genome-wide injection11Identify gene function byIdentify gene function byinsertional insertional or chemical mutagenesisor chemical mutagenesis1) T-DNA or transposon insertionsand PCR-based screens2) Arabidopsis Tilling project12 Gene XNPTIIFRLBRBPCR products:1. Screen for T-DNA (or Ds) insertion in specific genesScreening pools (p1-p5) p1 p2 p3 p4 p5 1kb ladder p1 p2 p p4 p5 LB/F RB/R13Data-base searches for T-DNA insertions in the genes of interests Gene XLBRBEnzyme digestionLigasePCR SequencedatabaseSalk Institute Genomic Group (http://signal.salk.edu/cgi-bin/tdnaexpress)LEUNIG="At4g32550SEU=At1g43850142. TILLING (Targeting Induced Local Lesions IN Genomes)Arabidopsis Tilling website:http://tilling.fhcrc.org:9366/EMSIsolated DNApoolGene-specific primersPCRheat & coolCEL IDenature1 2 3 4 5 6 7 8 9 1015Homologouse recombination and gene knock-out in yeast and mouse (Page 744-746, 852, A.7; A.8; E.8)16Fig. A.817Fig. A.718Fig.
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