Lecture_C.elegans_handouts

Lecture_C.elegans_handouts - IV. Caenorhabditis elegans...

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1 IV. IV. Caenorhabditis Caenorhabditis elegans elegans Caenorhabditis Caenorhabditis elegans elegans as a as a model model organism organism • One of the simpliest organisms that has several traits of interest • Easy to manipulate – – Small – Grown in Petri dishes – Three day generation time – Strains can be kept as frozen stocks • Transparent and many abnormalities can be seen under light microscope
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2
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3 A. Forward Genetic Screens
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4 Phenotypes Phenotypes - examples examples Egg-laying defective Mutation in transcription factors tbx-8 Mutation in transcription factors controlling differentiation of muscle http://www.wormbook.org/chapters/www_RTK RasMAPKsignaling/RTKRasMAPKsignaling.ht ml http://genomebiology.com/2005/6/5/R45 http://genomebiology.com/2005/6/5/R45 1. 1. Simple Simple screens screens • Routinely used in worm laboratories • Can identify genes involved in a specific process • Allows to identify visible, recessive mutations • In a typical screen with 12,000 haploid genomes we expect to recover 6 mutations in particular gene (1 mutation every 2000 copies)
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5 1. 1. Simple Simple screens screens Mutagen is used to induce mutation in the sperm and oocytes of wild type hemaphrodytes Mutagenized worms are grown for two generations to produce homozygous mutants Worms showing mutant phenotype are separated to determine if their phenotype is transmitted to next generation Requires every animal to be inspected for the presence or absence of particular phenotype Phenotype is not always informative or revealing in which pathway the mutagenesis occured Mutagenized animals Wild type Heterozygous (mut/+) Wild type Homozygous (+/+) Heterozygous (mut/+) Homozygous (mut/mut) Simple Simple F 2 screen screen 1) Hemaphrodite is exposed to a mutagen and genes are randomly mutated in the germ cells 2) Fertilization of an egg by mutated sperm result in heterozygous (mut/+) F 1 3) ¼ of F 2 is homozygous for the mutation and result in a phenotype 4) Mutant animal is grown for several days and F3 is inspected to determine whether the mutant phenotype breeds through 1 3 4 2
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6 Mapping the mutation Mapping the mutation • Identyfying the molecular nature of mutated gene relies on three criteria: – Consistent map position – Sequenced mutation – Rescue of phenotype – DNA can be injected to the oocyte to form a large, stable molecules called extrachromosomal arrays • RNAi is very useful in C. elegans screens • Background of endogenous transposons makes it very difficult to use transposon tagging of mutated gene • Worms can be randomly mutated and kept as a libraries Identifying Identifying genetic genetic patwyas patwyas • Once the phenotype associated with particular process is found, more genes involved in the same process may be found by: – Simple screens – simply looking for more mutants with the same phenotype – Modifier screens
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7 1. 1.
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Lecture_C.elegans_handouts - IV. Caenorhabditis elegans...

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