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Mouse as a Model Organism

Mouse as a Model Organism. Tuesday, February 7, 2012. Overview. Reproduction Grafting Non-Homologous Recombination Homologous Recombination Cre / loxP Recombination Tissue Growth Histology Models of Human Disease. Reproduction. 5-10 litters / year 5-10 pups / litter

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Mouse as a Model Organism

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  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

  8. 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

  14. 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

  15. Homologous recombination

  16. BMP7

  17. 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.

  18. Cre / loxP Recombination

  19. 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

  20. 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

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

  22. 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 TUNEL Nile Blue Acridine Orange

  23. 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.

  24. 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?

  25. Comparison of Vertebrate Models

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