Von Mäusen und Menschen E - 1

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1 Von Mäusen und Menschen E - 1

2 Mus musculus: Genetic Portrait of the House Mouse E - 3 Outline Mouse genome Mouse life cycle Transgenic protocols Addition of genes by nuclear injection Removal of genes by targeted mutagenesis Uses of transgenic technology to determine Function of gene products Characterizing regulatory regions Links between mutant phenotypes and transcription units Creating a mouse model for human disease E - 4

3 The Mouse Genome Similar to humans Contains about 3 billion nucleotides Homologues for every gene in humans Differences due to species-specific additions to gene families 19 autosomes and 2 sex chromosomes Conserved synteny Genes closely linked in mouse are also closely linked in humans Average size of conserved syntenic regions: 17.6 Mb Since common ancestry, human and mouse genomes have broken apart and rearranged ~ 170 times E - 5 Comparison of Mice and Humans Trait Average weight Average length Genome size Haploid gene number Number of chromosomes Gestation period Age at puberty Estrus cycle Life span Mice 30g 10 cm (without tail) 3,000,000,000 bp 25, autosomes + X and Y 3 weeks 5-6 weeks 4 days 2 years Humans 77,000 g (170 lb) 175 cm 3,000,000,000 bp 25, autosomes + X and Y 38 weeks weeks 28 days 78 years E - 6

4 Conserved Synteny Fig. E.3 E - 7 Life Cycle Fig. E.4 E - 8

5 Germ Cell Development Female Born with all gametes they will ever have ~ 50,000 eggs, or oocytes Oogenesis begins in ovaries of fetus Oogonia enter meiosis but stop at diplotene in first prophase Estrus cycle begins at puberty 4 days in mice 8-10 primary oocytes complete first meiotic division Secondary oocyte stops at metaphase are released from ovary (ovulation) Oocyte passed into oviduct and is receptive to fertilization (estrus) E - 9 Germ Cell Development Males Sperm At puberty, large number of haploid gametes produced for rest of life Spermatogenesis E - 10

6 Figure 4.18 E - 11 Fertilization E - 12

7 Early Embryo Development Preimplantation starts at conception 22 hrs sperm head expands into pronucleus Next 60 hours - embryo divides four times equally (totipotent) cleavage stage Twins and Chimeras E - 13 Events Restricting the Developmental Potency 16-cell embryo First differentiation events occur Trophectoderm layer cells on outside of embryo Inner cell mass (ICM) cells on inside of embryo Fetus is derived from ICM Blastocyte forms After implantation, placenta develops, embryo grows, and tissues and organs emerge E - 14

8 Two Powerful Transgenic Techniques Addition of genes by nuclear injection Foreign DNA injected into pronucleus of fertilized egg Place injected one-cell embryo back into oviduct DNA integrates at random into chromosome (25-50% of cases) Targeted mutagenesis (knock-out mice) Transfection of cultured embryonic stem cells. Homologous recombination may occur after transfection Injection of transformed cells back into blastocoele Some cells integrate into germ line E - 15 How transgenic Mice are created Fig. E.7 E - 16

9 Knocking Out a Gene in ES Cells E - 17 Using Transgenic Tools Determination of gene function Combining mouse gene with regulatory regions from other mouse gene Expression of transgene can be changed: At higher than normal level In an alternative tissue At alternative developmental stage E - 18

10 Using Transgenic Tools SRY locus responsible for production of maleness E - 19 Application of Transgenic Technology Transgenic expression of myc gene provides information on gene s role in tumor formation (a) structure of gene (b) northern blot analysis E - 20

11 Using Transgenic Technology to Characterize Regulatory Regions DNA construct containing mouse regulatory region of interest is attached to lacz reporter gene Fig. E.11 E - 21 Mapping of cis-acting regulatory region of Tcp 10b t gene Fig. E.12 E - 22

12 Transgenic technology to identify locus responsible for mutant phenotype Dominant deletion mutation at T locus causes short tail Transgene mouse with pme75 transgene mated to mutant Normal phenotype demonstrates deletion of pme75 is cause of short tail Fig. E.13 E

13 Targeted Mutagenesis to Create a Mouse Model for Human Disease E - 25 E - 26

14 E - 27 E - 28

15 E - 29 Inheritance Pattern E - 30

16 Binary transgenic Mice VM VL Musculus-31 Tetracyclin-Tetracyclin induced Gene expression VM VL Musculus-32

17 VM VL Musculus-33 Reverse Tet-System VM VL Musculus-34

18 Dual Promotor with reporter gene VM VL Musculus-35 Inducible Expression using the Gal-System VM VL Musculus-36

19 Inducible Expression using the Gal-System VM VL Musculus-37

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