Transgenic Mouse Models Bio 426 / HS 2012

1. Transgenic Mouse ModelsBio 426 / HS 2012 Kurt Bürki, Pawel Pelczar Institute of Laboratory Animal Science, University of Zurich

2. Goals • To cover the techniques to generate transgenic models • To compare advantages / disadvantages / limits of the techniques • To discuss important models in several fields of biomedical research

3. Learning Objectives By the end of the lecture series the participants are able to: • List advantages and disadvantages of the major methods to generate transgenic animals • Design functional transgenes and targeting vectors • Present and critically discuss original papers in the field in a comprehensive form (key skill)

4. Additional Practical Courses • BIO 413: Generation of Transgenic Animals (LTK Module 3E) • BIO 412: Einführung in die Labortierkunde / Introduction into Laboratory Animal Science (LTK Modul 1)

5. Structure of the Lecture Series • Introduction • Technical Aspects (Students: Paper to read / Comrehensive presentation) • Transgenic Mouse Models (Students: Paper or review to read / Identification of questions relevant for a given field) • Exam • Visit of a Laboratory

6. Transgenic Animals:Definition Mutant animals carrying experimentally introduced foreign genetic elements in all their cells, including the germline

7. Steps towards a Transgenic Model • Working hypothesis • Gene Construct • Insertion into an early embryonic stage • Screening for transgenic animals • Profiling of expression pattern • Phenotyping • Model Validation / Experimentation

8. Gene Construct • Expression constructs (transgenes) • Viral vectors: retroviral/lentiviral vectors • Targeting constructs: comprising homologies to murine sequences

9. Gene Insertion • Insertion by nuclear DNA repair / recombination mechanisms • Random (non-homologous end joining NHEJ: subject to position effects) • Targeted (homologous recombination)

10. The Mouse genome

11. Genes Phenotype Genetic Networks

14. Transgene Promoter/Coding Sequence Insertion Site Targeting Vector Knock-out/Knock-in Conditional Mutants Phenotype Loci, Genes Position Effects Variegated Expression Penetrance Expressivity Polygenic Traits Genetic Background Phenotype Transgenics vs. Genetics

15. The Mouse as an Experimental System Life Cycle 4-day oestrus 20-day gestation 4-8 pups per litter 2-8 litters per female 7 weeks to sexual maturity 2-3 year lifespan Genome Mammalian 20 chromosomes 2.6 Gb ~25000 genes 99% have human counterpart Reverse genetics Knockouts Transfenics Conditional expression Inducible expression Retroviral vectors siRNA Strains Inbred Outbred Recombinant inbred Consomic Fluorescent Tools Genome sequence Embryonic stem cells Expression arrays Gene-trap libraries Insertional vector libraries BAC libraries Assisted reproduction Cryopreservation Embryo rederivation In vitro fertilization Intracytoplasmic sperm injection Cloning

16. Why the Mouse? Of the model organisms which may be genetically modified, the mouse is: • The closest to humans– • mammal • The most complex - • integration of systems (endocrine, immune, nervous etc.) • Genetic manipulation is extremely versatile– • Gain-of-Function (Transgenesis), Loss-of-Function • (knock-out), Change-of-Function (knock-in); • temporally and spatially restricted (conditional)

17. Applications of transgenic mice Transgenic mice are often generated to address the role a gene plays in a biological process at the level of the whole organism: - To confirm the role of a gene mutation - To help unravel the molecular mechanisms that control gene expression - To help unravel the biochemical in vivo mechanisms and the origin of disease - To develop an animal model to test therapeutic strategies

18. Transgenic Animals: Methods • Classical • - Pronuclear Microinjection • - Lentiviral Infection • - Embryonic Stem (ES) Cell Gene Transfer • - ES Cell mediated Gene Targeting (knock-out, knock-in) • Experimental • - Transfection of Somatic Cells - Cloning • - Sperm Based Transfection (ICSI) • - Transposons

19. Generation of transgenic animals

20. pros Relatively simple and efficient Long transgenes possible Potentially all species Very efficient Single copy insertions No technical equipment Works in many species Long transgenes possible Gene targeting possible Single copy insertions cons Random integration Multicopy insertions ( Strain limitations) High embryo mortality 9.5 kb packaging limit Safety issues (?) Only random integration Technically difficult Time consuming Species / Strain limitations Mouse Transgenesis Methods Pronuclear microinjection Lentivral infection ES based transgenesis

21. Pronuclear Microinjection • Microinjection of DNA directly into the pronuclei of fertilized eggs • Implantation of the microinjected eggs into a surrogate mother • Allowing the embryos to develop to birth • Demonstrating that the foreign gene has been stably incorporated into the host genome and that it is heritable in at least one of the offspring • Demonstrating that the gene is expressed and regulated correctly in the host organism

22. Microinjection Station

23. Establishment of ES Cells in vitro Blastocysts d 3-4 ES-Cell-Colonies ICM (Innere Zellmasse)

24. Germline male chimera (C57BL/6 in BALB/c) with offspring

25. Timeline: Transgenesis by Pronuclear Microinjection or Lentiviral transfection DNA or LV injection Mate founders Identyfy founders Begin analysis Birth 0 2 4 6 8 10 12 gestation gestation maturation of founders maturation of F1 progeny

26. Timeline: generation of ES cell-derived mice Identify homologous recombinants by DNA analysis Identify mouse Chimeras with high ES cell contribution Introduce targeting vector into ES cells Begin analysis Germline transmission 0 2 4 6 8 10 12 gestation gestation Drug selection Colony growth and expansion Inject clones into blastocysts Sexual maturation of chimeras Identify male and female heterozygotes Sexual maturation of heterozygotes Identify homozygotes

27. Trends in the Field of Transgenic Animals • More Refined Transgene Systems: • - temporal regulation (tet ON/OFF) • - tissue specific and temporal regulation ( Cre/lox) • Gene Targeting in Species other than the Mouse • IntegrativeDatabases • Animal Welfare Aspects

28. Trends with Transgenic Animals (1) Targeted Modifications, Control over Expression or Silencing, Combined (binary) Systems • Inducible Transgene-Expression Tet-on, Tet-off Systems) • Tissue-specific knock-outs (Cre-lox System) • Inducible knock-outs (CreMER System)

29. Trends with Transgenic Animals (2) • Routine Gene-Targeting in Mammalian Species other than the Mouse • New: Gene Targeting in Rat ES Cells / iPS Cells / Spermatogonial Stem Cells • New: Zinc-Finger Nucleases for the Introduction of Site- Directed Genome Modifications

30. Transgenic Animals: Potential Problems • Technical problems to closely mimic a desired situation • Underestimation of biological complexity • Mouse – Human differences • Inappropriate analysis • Undefined genetic backgrounds

31. Example: The App Gene (Alzheimers Disease)

32. Paper to Read Brinster R.L. et al.: Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci USA 82, 4438-4442 (1985).