BIOC454-2011-MBouchard-Transgenics

BIOC454-2011-MBouchard-Transgenics - BIOC-454 Transgenesis...

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BIOC-454 Transgenesis, Gene targeting and Cloning Maxime Bouchard Goodman Cancer Research Centre Rm 415 [email protected]
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Topics covered • Overview of the mouse as a model system • Transgenic mouse generation • Gene targeting/chromosome engineering • Cloning Goal: give an overview of genome alteration strategies in the mouse used to study gene/genome function
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The MOUSE Life span: approx. 2.5 years Gestation : 21 days Litter size: 6 to 12 Generation time: two-three months Several inbred and outbred strains Genomic/gene expression databases available Most advanced genetic technologies Cost per mouse: $1 to 10$/day Mouse and human genomes are highly conserved
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Kerstin Lindblad-Toh Whitehead/MIT Center for Genome Research
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Is the mouse relevant to study human disease? ->Human/mouse genomic comparison Human and mouse both have about 30,000 protein-coding genes. Mouse genes without human homologue < 1% 90% of the human and mouse genomes are syntenic Segments in which gene order from most-recent common ancestor has been conserved 40% of both genomes can be aligned at the nucleotide level (rest is mostly repetitive elements) Protein-coding sequences represent 1.5% of mouse genome Source: Mouse genome sequencing consortium, Nature 420:520 (2002)
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Types of Animal Disease Models Human disease gene inactivated in the mouse (or other model organism) May or may not resemble human disease Manipulate mouse genome to recreate complex human disease Insert exogenous chromosome segments Delete large chromosomal regions (MICER technology) Mouse KO or transgenic resembles human disease (fortuitous discovery) “Home made” knockout or transgenic mice Genetrap project Mouse KO or transgenic does not immediately resemble disease Model-to-be Informative for tissue development/physiology
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Mutagenesis Transgenesis Gene targeting Manipulating the mouse genome Nuclear transfer (cloning)
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Transgenesis
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1982 First transgenic mouse Richard Palmiter and Ralph Brinster Metal-responsive promoter (MT) Growth Hormone (human) Exogenous zinc Initial goal was to rescue “little” mutation (defcient in growth hormone)
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Basic Transgene design Temporal and spatial expression Intron Gene of interest AATAAA Transgene <1kb to >200kb (Intron passes mRNA through splicing machinery which stabilizes transcript => better expression)
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Promoter cDNA Gain of Function 1- Tissue specifc (brain, liver, muscle …) 2- Ubiquitous 3- Inducible (tetracyclin, interFeron…) Loss of Function - wild-type gene - GO± mutant - dominant negative - siRNA - antisense - ribozyme Basic Transgene design Reporter - LacZ - G±P…
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Stable lines Transient
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Some interesting transgenic mice… Brinster Nagy - hPVR (human poliovirus receptor) transgenic (Ren et al., 1990): -> mice become susceptible to polio - Serotonergic (5-HT) receptor transgenic Engel et al. (1998) : -> mice increase alcohol consumption by 46% GFP-expressing transgenic mice
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BAC Recombineering BAC: Bacterial Artifcial Chromosome (150-200 Kb) oF genomic DNA Use for transgenic mouse generation (pronuclear injection)
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BIOC454-2011-MBouchard-Transgenics - BIOC-454 Transgenesis...

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