1. 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

2. 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 Figure 1.22 Kit gene

3. 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

4. Embryonic stem cells: blastocyst-stage cells (from inner cell mass) that have been coaxed into growing in culture blastocyst inner cell mass

5. Blastocyst stage cells can be easily incorporated into a different blastocyst stage embryo, allowing production of chimeric mice Fig. 8.26 mouse with 4 parents!! mom and dad have white fur mom and dad have black fur

6. A mouse with 3 of its parents (6 total!) Fig. 8.26

7. Adding a gene: Producing Transgenic Mice

8. 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

9. Production of Transgenic Mice

11. RNA Gene X RNA Gene X A normal cell has two copies of a gene (ie. BMP7) No RNA Gene X RNA Gene X Neo resistance gene 1.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.) 2.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. Recipe to "knockout" a gene: Mario Cappechi Oliver Smithies

12. Technique for Gene Targeting #1 #2 #3

13. Now you have heterozygous ES cells--how do you make a homozygous mutant mouse? #4 #5

14. Now you have a chimeric mouse… #6 #7

15. Sometimes the effects are dramatic! BMP7 knockoutWild-type

16. Morphological Analysis of Bmp7 Knockout Mice

17. Sometimes the effects are not dramatic --no phenotype!

18. Mouse models of human disease allow us to design and test new treatments Oliver Smithies CFTR and cystic fibrosis

19. Ultrabithorax mutant Wildtype remember me?

20. The Homeotic genes in Drosophila Fig 6.35 ANT-CBX-C

21. Ed Lewis had predicted that the homeotic genes would shape the body plans of all animals

22. In vertebrates the Hox genes have been duplicated, creating four clusters Figure 8.30

23. Different Hox genes are expressed at different places along the anterior-posterior body axis

24. Knocking out Hoxc8 Figure 8.30

25. Partial transformation of the first lumbar vertebra into a thoracic vertebra by knockout of the Hoxc8 gene

26. Genetic analysis of Hox genes is more complicated in mice paralog group Knocking out Hoxa10, Hoxc10 & Hoxd10 Figure 8.30

27. The duplication of the Hox clusters means that in the mouse, Hox genes work together to give each body region its own identity Lumbar vertebrae transformed to thoracic vertebrae Figure 8.32 wildtype Hoxa10 Hoxc10 Hoxd10 triple mutant

28. Remember the segment-polarity genes wingless and engrailed? Wg En

29. Retroviruses can also cause cancer by inserting next to and thus activating the expression of proto-oncogenes wnt-1 gene exons Transcribe to mRNA 5 kilobases retroviral insertion sites in different tumors

30. Expresses Wnt-1 Expresses En-1 Wildtype brain Wnt-1, The mouse homolog of wingless, is normally expressed at the midbrain-hindbrain junction

31. Expresses Wnt-1 Expresses En-1 Structures lost in Wnt-1 mutant Wildtype brain Brain of Wnt-1 mutant

32. Rules of Evidence What type of experiment is this? Pax6

33. Normal eye Aniridia eye small eye mutant mouse Pax6 regulates eye development in flies, squid, mice, and us iris no iris

34. When eyeless (Pax6 homolog) is expressed at the ends of fly legs, extra eyes form there!

35. When squid Pax6 homolog is expressed at the ends of fly legs, also see extra eyes! ectopic eye (squid Pax6) ectopic eye (fly Pax6)

36. Wild-typeSplotch mutant The Pax-3 gene is altered in a classic mouse mutation

37. Mutations in Pax3 lead to Waardenburg Syndrome I. dominant mutation eyes can be different colors white patch of hair (forelock) deafness

38. 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

39. White spotting and Steel: Connecting classic mouse mutations to stem cells and cancer

40. An example of stem cells: the blood cell lineage

41. Cells lacking signal behave differently than cells lacking receptor + + + mutant + + +++ ++ +++ + ++++++++ + + If mutant cells lack signal, they can be rescued by wildtype neighbors which make signal. If mutant cells lack receptor, they cannot be rescued by wildtype neighbors which make signal. + + + mutant +++ ++ ++ + ++++++ +++ ++++ Thanks, I needed that! What? I can't hear you! Mosaics can help us understand gene and thus protein function

42. White-spotting and Steel: Which is signal and which is receptor?? The mutant blood cells migrated to the bone marrow. Experiment #1 Put blood cells from Steel homozygous mutant embryos into a wild-type host. 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

43. Steel encodes a diffusible ligand and White-spotting (Kit) encodes its transmembrane receptor tyrosine kinase domain activated when Steel binds, phosphorylating target proteins Fig. 3.22 melanin genes PAX3 activates Mitf

44. Mutations in MITF lead to Waardenburg Syndrome II.