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Model organisms: mice. vertebrates! mice are ~ 3 inches long, can keep many mice in a room. generation time is ~ 3 months, so genetics can be done history - scientists have worked with mice for 100 years
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Model organisms: mice • vertebrates! • mice are ~ 3 inches long, can keep many mice in a room. • generation time is ~ 3 months, so genetics can be done • history - scientists have worked with mice for 100 years • genetic tools - can introduce extra genes or remove a specific gene, then study the effect on development • Disadvantages: development inside the mother, hard to see. Expensive! Large Genome = 3 Gb
The mouse provides a superb model for human development and disease because we share virtually ALL of our genes and use them in similar ways Kit gene Figure 1.22
Genetic analysis: creating transgenic mice Problem: Find a cell line that can grow in tissue culture but also retains the potential to become part of a real embryo. Solution:Embryonic stem cells
blastocyst inner cell mass Embryonic stem cells: blastocyst-stage cells (from inner cell mass) that have been coaxed into growing in culture
Blastocyst stage cells can be easily incorporated into a different blastocyst stage embryo, allowing production of chimeric mice mom and dad have black fur mom and dad have white fur mouse with 4 parents!! Fig. 8.26
A mouse with 3 of its parents (6 total!) Fig. 8.26
Production of Transgenic Mice Embryonic stem cells (ES cells) are then incorporated into blastocysts, with the hope that they “go germline”. If so, a line is created
Recipe to "knockout" a gene: A normal cell has two copies of a gene (ie. BMP7) RNA Gene X RNA Mario Cappechi Gene X Insert gene for resistance to the drug neomycin into the middle of gene X, destroying its function. (Gene X is contained in a DNA plasmid.) Introduce gene X KO plasmid into ES cells and use homologous recombination to replace one of the wildtype copies of gene X with mutant gene. Neo resistance gene No RNA Gene X Oliver Smithies RNA Gene X
Technique for Gene Targeting #2 #1 #3
Now you have heterozygous ES cells--how do you make a homozygous mutant mouse? #4 #5
Sometimes the effects are dramatic! Wild-type BMP7 knockout
Sometimes the effects are not dramatic --no phenotype!
Mouse models of human disease allow us to design and test new treatments CFTR and cystic fibrosis Oliver Smithies
remember me? Wildtype Ultrabithorax mutant
The Homeotic genes in Drosophila ANT-C BX-C Fig 6.35
Ed Lewis had predicted that the homeotic genes would shape the body plans of all animals
In vertebrates the Hox genes have been duplicated, creating four clusters Figure 8.30
Different Hox genes are expressed at different places along the anterior-posterior body axis
Knocking out Hoxc8 Figure 8.30
Partial transformation of the first lumbar vertebra into a thoracic vertebra by knockout of the Hoxc8 gene
Genetic analysis of Hox genes is more complicated in mice Knocking out Hoxa10, Hoxc10 & Hoxd10 paralog group Figure 8.30
The duplication of the Hox clusters means that in the mouse, Hox genes work together to give each body region its own identity wildtype Hoxa10 Hoxc10 Hoxd10 triple mutant Figure 8.32 Lumbar vertebrae transformed to thoracic vertebrae
Remember the segment-polarity genes wingless and engrailed? Wg En
Retroviruses can also cause cancer by inserting next to and thus activating the expression of proto-oncogenes retroviral insertion sites in different tumors Transcribe to mRNA 5 kilobases exons wnt-1 gene
Wnt-1, The mouse homolog of wingless, is normally expressed at the midbrain-hindbrain junction Expresses Wnt-1 Expresses En-1 Wildtype brain
Structures lost in Wnt-1 mutant Expresses Wnt-1 Expresses En-1 Wildtype brain Brain of Wnt-1 mutant
Pax6 Rules of Evidence What type of experiment is this?
Pax6 regulates eye development in flies, squid, mice, and us iris Normal eye no iris small eye mutant mouse Aniridia eye
When eyeless (Pax6 homolog) is expressed at the ends of fly legs, extra eyes form there!
When squidPax6 homolog is expressed at the ends of fly legs, also see extra eyes! ectopic eye (fly Pax6) ectopic eye (squid Pax6)
The Pax-3 gene is altered in a classic mouse mutation Wild-type Splotch mutant
Mutations in Pax3lead to Waardenburg Syndrome I. • dominant mutation • eyes can be different colors • white patch of hair (forelock) • deafness
Why Models Matter The Example of Mutation of the Kit gene in humans and mice • “Piebaldism” • Affected individuals are anemic, sterile, deaf, and lack pigment in certain skin cells • Kit encodes a receptor tyrosine kinase and is required for cell proliferation in neural crest, blood, and germ cells Figure 1.22
White spotting and Steel: Connecting classic mouse mutations to stem cells and cancer
An example of stem cells: the blood cell lineage
+ + + + + + + + + mutant + mutant + + mutant + + + + + + + + + + + + + + + + + + mutant + mutant + + mutant + + + + + + + + + Cells lacking signal behave differently than cells lacking receptor Thanks, I needed that! Mosaics can help us understand gene and thus protein function + + If mutant cells lack signal, they can be rescued by wildtype + neighbors which make signal. What? I can't hear you! + If mutant cells lack receptor, + they cannot be rescued by wildtype + neighbors which make signal.
White-spotting and Steel: Which is signal and which is receptor?? Experiment #1 Put blood cells from Steel homozygous mutant embryos into a wild-type host. The mutant blood cells migrated to the bone marrow. Experiment #2 Put blood cells from White-spotting homozygous mutant embryos into a wild-type host. These mutant blood cells did not migrate to the bone marrow. Steel is the Signal- Mutant cells can still receive information White-spotting is the receptor- Mutant cells cannot receive information
Steel encodes a diffusible ligand and White-spotting (Kit) encodes its transmembrane receptor PAX3 activates Mitf tyrosine kinase domain activated when Steel binds, phosphorylating target proteins melanin genes Fig. 3.22