Functional and Biomedical Aspects of Genome Research

20 11 35 Vorlesung SS 04 Bartsch, Jockusch & Schmitt-John

Mi. 9:15-10:00, in W7-135

5. Vertebrate Models for Functional Genomics: A lot of Fish, some Mouse

Thomas Schmitt-John

Mosquito (Anopheles gambiae)

Fruit Fly (Drosophila melanogaster)

Nematodes (Caenorhabditis elegans andCaenorhabditis briggsae)

Invertebrate Animal Models

Zebrafish (Danio rerio)

Vertebrate Animal Models

Frog (Xenopus laevis)

Chicken (Gallus gallus)

Rat (Rattus norvegicus)

Mouse (Mus musculus)

Chimpanzee (Pan troglodytes)

Pufferfish (Fugu rubripes)

www.ensembl.org

Organism Sequence [bp] Transcripts Chrs.State 2004 State 2004

Human 3.201.762.515 31.609 22, x, y Chimpanzee ~2.730.000.000 ? 23, x, yMouse 3.167.465.022 34.076 19, x, y Rat 2.571.104.688 30.232 20, xChicken ? ? 32, w, zZebrafish 1.464.723.148 30.783 25Fugu 329.140.338 38.510 22 Mosquito 278.253.050 18.634 4, xFruit Fly 128.343.463 18.289 5, xC.elegans 103.006.709 22.941 5, xC.briggsae 105.835.413 14.713 5, x

Present State of Genome Projects

Zebrafish Genome: Present StateLast Update: 08-02-2004 Ensembl gene predictions: 22.409 Genscan gene predictions: 55.567 Ensembl gene exons: 200.207 Ensembl gene transcripts: 30.783 Contigs: 62.895 Clones: 62.895 Base Pairs: 1.464.723.148 Golden Path Length: 1.459.115.486

The Zebrafish (Danio rerio):25 ChromosomesLifecycle: About 3 monthsEmbryonic development: 3 Days

Properties of the Modelorganism Zebrafish

short lifecycle (3 months)small sizegynogenesistransparent extracorporal embryonic development

transgenic technologiescryopreservation

ZebrafishChromosomes

Zebrafish Development: 1 to 8 hours

10 to 20 hours

Zebrafish Development: 10 to 15 hours

Zebrafish Development: 16 to 21 hours

Zebrafish Development: 22 to 28 hours

Zebrafish Development: 48 hours

Zebrafish Mutagenesis: Bodyshape Mutants

Brand et al.: Development. 1996 Dec;123:129-42.

Zebrafish Mutagenesis: Head and Eye Mutants

Brand et al.: Development. 1996 Dec;123:129-42.

Zebrafish Mutagenesis: Optic Chiasm

Brand et al.: Development. 1996 Dec;123:129-42.

Imai Y, Feldman B, Schier AF, Talbot WS. Genetics. 2000 155:261-72.

Zebrafish Mutagenesis: Cyclopedia Mutationmapped to oep on LG10

Imai Y, Feldman B, Schier AF, Talbot WS. Genetics. 2000 155:261-72.

Zebrafish Mutagenesis: Cyclopedia Mutationmapped to distal LG12

Identification of Mutated Genes by Positional Cloning

assessment and verificationof the phenotype

segregation mapping identification ofpositional candidates

P608 K609 S610 P606 E609S610

ACT GAG GCA GAA GGT GAA GGA GAG GAA GCA GAA GCT AAG GAG GAA AAG AAA ATT GAG GGA AAG GTT GAG GAA GTG GCT GTC AAG GAG GAA ATC AAG GTC GAG AAG CCT GAG AAA GCC AAA TCC CCT ATG CCC AAA TCA CCC GTG GAA GAA GTA AAG CCA AAA CCA GAG GCC AAG GCC GGG AAG GGT GAG CAC AAG GAG GAA GAG AAA GTT GAG GAA GAG AAG AAG GAA GTC ACC AAA GAATCA CCC AAG GAA GAG AAG GTG GAG AAA AAG GAG GAG AAG CCA AAA GAT GTT GCA GAT AAA AAG AAG GCC GAG TCC CCG GTG AAA GAG AAG GCT GTG GAG GAG GTG ATC ACC ATC AGC AAG TCG GTA AAG GTG AGC CTG GAG AAA GAC ACC AAA GAG GAG AAG CTG CAG CCG CAG GAG AAG GTG AAG GAG AAG GCA GAG GAG GAG GGG GGC AGT GAG GAG GAA GGG AGT GAC CGA AGC CCG CAG GAG TCC AAG AAG GAA GAC ATA GCT ATC AAT GGG GAG GTG GAA GGA AAA GAG GAG GAG GAG CAG GAA ACT CAG GAG AAG GGC AGT GGG CGG GAG GAG GAG AAA GGG GTA GTC ACT AAT GGC TTA GAT GTG AGC CCT GCA GAA GAG AAG AAA GGA GAG GAT AGC AGT GAT GAT AAA GTG GTG GTC ACC AAG AAG GTA GAA AAG ATC ACC AGC GAG GGG GGC GAT GGT GCT ACC AAA TAC ATC ACCAAA TCT GTA ACC GTC ACT CAA AAG GTT GAA GAG CAT GAG GAG ACC TTT GAG GAG AAG CTG GTC TCA ACT AAA AAG GTA GAA AAG GTC ACT TCA CAC GCC ATA GTC AAG GAA GTC ACC CAG GGT GAC TAA GATCGGAGTCCGTTGCAAAAGGT

sequencing ofpositional candidatesby mutant and wildtype

identification of the causative mutation

Mouse Genome: Present StateLast Update: 08-02-2004 Ensembl gene predictions: 26.762 (incl. 1455 pseudogenes)Genscan gene predictions: 100.246 Ensembl gene exons: 219.510 Ensembl gene transcripts: 34.076 Contigs: 128.285 Clones: 86.934 Base Pairs: 3.167.465.022 Golden Path Length: 2.627.444.668

The Mouse (Mus musculus):19 Autosomes and X and Y ChromosomesLifespan: About 2 YearsSexual fertile: 6 to 8 weeksPrenatal development: 21 DaysInbred strains available

mutant mouse

zygote ES cells, homologous recombination screen in culture

ENUpronucleus injection gene trapping knock out (KO)

knock in (KI)spontaneousradiation etc.

F1: dominantF2/F3: recessive

F1: transgenic mouse

F1: heterozygous KO/KIF2/F3: homozygous KO/KI

chimera

random gene specific,site random

random gene and site specific

physical and chemical mutagens DNA mutagenesis

mating foster mother

Mutagenesis of the Mouse

Origin Type Background Phenotype Reference:

UNC Knockout outbred severe intestinal disease Snouwaert et al.Edinburgh Partial knockout Mixed? v. mild Dorin et al.Cambridge Knockout Mixed? Severe Ratcliff et al.Baylor Knockout C57BL Severe O'Neal et al.Baylor Knockout Severe Hasty et al.Cambridge D F508 Severe Colledge et al.Utah D F508 C57BL Severe Zeiher et al.Rotterdam D F508 Severe van DoorninckBrisbane G551D Less severe intestinal block Delaney et al.Toronto Knockout C57BL/6J Severe, + lung disease Kent et al.Toronto Knockout several varied by background Rozmahel et al.

Disease Model Mice for Cystic Fibrosis (CF)

Disease Model Mice for Sickle-Cell Anemia

Sickle-shaped erythrocytesof transgenic mice expressing mutant ß-hemoglobin

Sickle-shaped erythrocytesclogging in capillariesScience 1997, volume 278, page 873

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WILDTYP, +/? WOBBLER, wr/wr

The wobbler-Mouse, a Disease Model forAmyotrophic Lateral Sclerosis (ALS) and Globozoospermia

SP