1. Mouse as a Model Organism Tuesday, February 7, 2012

2. Overview Reproduction Grafting Non-Homologous Recombination Homologous Recombination Cre / loxP Recombination Tissue Growth Histology Models of Human Disease

3. Reproduction 5-10 litters / year 5-10 pups / litter 19-21 day gestation Sexually mature at 7 weeks 4-5 generations per year

4. Grafting Cannot do it! Cells are too small

5. Techniques in Mouse

6. 3 Types of Genetic Modifications Insertion – of a transgene or a modified allele, i.e., “knock-in” – can produce a gain of function mutation Knockout – of a particular gene or piece of DNA – to assess a gene’s function, i.e., is it necessary for a particular role in development Conditional Mutant – a spatially and temporally specific knockout!

7. Creating transgenic mice

9. I. Inserting DNA into Cells 1) Microinjection of cloned gene into nucleus of newly fertilized egg 2) Transfection incubate ES cells in solution that makes them take up the DNA, very inefficient need to identify cells that took up the DNA with reporter such as drug resistance 3) Electroporation – a high voltage pulse “pushes” DNA into cells 4) Retroviral vectors – a more natural way or getting genes into cells

10. Microinjection

11. Transfection

12. Electroporation Highly efficient for the introduction of genes in mammalian tissue culture cells

13. Retroviral vector

15. II. Knocking out a gene Homologous recombination – Clone gene that is nonfunctional – Introduce DNA into cell by any method discussed above – Homologous recombination will occur replacing endogenous gene with nonfunctional gene

16. Homologous recombination

17. BMP7

18. Conditional Mutant: Cre-LoxP Conditional mutants are needed when you want to study the effects of a gene in certain tissue late in development but the gene is also necessary early in development. A traditional knockout would result in a mutant that does not develop to stage needed. Cre is a recombinase that excises DNA located in between LoxP sites You generate two transgenic lines one that expresses Cre in the tissue you are interested and a second that contains gene of interest flanked by loxP sites. The gene will only be deleted where Cre is expressed. – Can also activate genes: In second line place stop signal flanked by loxP between 5’ regulatory element and gene. When stop signal is removed gene will be activated.

19. Cre / loxP Recombination

21. Tissue Culture Possible to grow certain tissues in vitro Need to have isolated stem cell line Most tissue type has different protocols Does not form functioning organ

22. Embryo and Organ culture Can remove entire embryo or organ and maintain alive in culture for a short period of time – Add factors to embryo or organ: activators, inhibitors, drugs Afterwards do whole mount or sections in situ hybridization, RT-PCR, immunostaining ect. to analyze the embryo or organ. Can also do tissue transplantations Can also remove at different stages to observe development

23. Histology Immunohistochemistry (Antibody staining) In situ hybridization Cell death staining Bone and cartilage chemical stains

24. Cell Death Staining TUNEL ( Terminal deoxynucleotidyl transferase dUTP nick end labeling), Nile Blue, Acridine Orange Used to detect apoptosis Tunel: detects DNA fragmentation by labeling the ends of the DNA Acridine Orange Nile Blue TUNEL

25. How to detect Cell Proliferation BrdU is a synthetic nucleoside that is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues. BrdU labeling: (Bromodeoxyurdine) BrdU incorporated into cells that are undergoing DNA synthesis. Detected with antibody staining.

26. Model of Human Disease Many known gene mutations exist that reproduce human diseases in mice. Are these accurate models of human disease? – Not all mouse phenotypes correspond to human phenotypes Studies primarily done in C57BL/6 strain – Is a study of a single strain sufficient to make conclusions about humans?

27. Comparison of Vertebrate Models MouseChickZebrafishXenopus Numbers of Eggs per ovulation 5-10 Development controlled by temp Lots and lots Initial Size~100 µm2-3mm600 µm~1 mm Gestation time19-21 days~20 days~1-2 days4 days to swimming tadpole Development Environment In uteroIn ovoExternal GenomeSequenced Genomic Manipulation CommonCannot Do ItCommonDifficult Surgical Manipulation Cannot Do It CommonCannot Do ItCommon