Mouse Models of Human Disorders

1. Mouse Models of Human Disorders Prepared by Rohan Palmer Institute of Behavioral Genetics University of Colorado at Boulder Rohan.Palmer@Colorado.edu

2. Goals • Understanding Genetic Influences on a Trait • Accounting for Variation in an Observed Trait • Why have animal models? • Response to Selection Study • Types of Mouse Models of Human Disorders • Transgenic Models • Mutagenic Models

3. Lecture Goal – Exam Question You are reading a Times articlewith a friend and you come across the following statement: “The study hypothesized that some of these susceptibility factors may be allelic variants of genes that govern embryonic serotonin neuron development and that these alleles may contribute to behavioral disorders by adversely increasing or decreasing serotonin system activity”. The article is about finding ways to create mouse models for human DCG’s. Your classmate has not yet taken Dr. Carey’s Behavioral Genetics course but fortunately you have. Describe to him/her what an allelic variant is and at how it might affect a behavioral disorder. Lastly, your friend looks at you as if you were crazy and says, “How does a mouse study, help US?” Based on what you have taken away from this course, convince your friend about the relevance and validity of mouse models. (20 Points)

4. Heritability The extent to which variation on a trait in a given population at a given point in time is attributable to genetic variation between individuals. VP = VG + VE h2 = VG/VP

5. Comparison Terminologies Synteny: The co-localization of genes on chromosomes of related species. Homolog: The situation where nucleic acid or protein sequences are similar because they have a common evolutionary origin. Often used loosely to indicate that sequences are very similar. Ortholog – gene sequences are similar between species. Paralog – gene sequences are similar within a species. Adapted from the Department of Energy; genomics.energy.gov

6. Why Create Animal Models? Pros • Test many hypothesizes • No human genetics ethical dilemma • Shortens length of study (Avg. gestational period is 19 days) (Females become fertile in 3 weeks) (Approximately 40 days for turnover; giving approximately 365/40 generations annually) Cons • Creating a “representative” phenotype is difficult • Ethical dilemma (Animal Rights; Institutional Animal Care and Use Committee (IACUC) • Can be very expensive. • Difficult to replicate.

8. Mouse Genome Informatics

9. Determining Genetic Influences on a Trait Response to 30 generations of selection for Open-Field Activity in Lab Mice (DeFries et al. 1978) Strain – A collective of animals from the same species that are genetically identical. Response to selection: DeFries et al. 1978 It is possible to observe variation within and between strains of mice. Variation on a measured trait within a stain is indicative of environmental influences on that trait. Variation on a measured trait between strains is indicative of genetic influences on that trait. For eg. The A/J inbred strain is susceptible to arthritis while the C57BL/6J strain is resistant. (Source: Adapted from Bioinformatics and Functional Genomics, p. 685) C57BL/6J mice consume far greater amounts of alcohol than DBA/2J mice.

10. Why does Emotionality in mice matter? • Humans • Includes anxiety and neuroticism • Think about what happens to you when you are anxious? • Rodents • Exploration, activity, anxiety, fear and emotionality/reactivity are measured in an open field apparatus (OFA)

11. Why does Emotionality in mice matter? • Humans • Includes anxiety and neuroticism • Think about what happens to you when you are anxious? • (Sweaty, elevated heart rate, preoccupied thoughts, urge to defecate, involuntary movement, Immobile) • Rodents • Low activity & high defecation scores define emotionality; measures are genetically correlated • Animals with high defecation scores are inactive

12. Balb B6 x F1 F1 x F2 F2 x F30 1st 10 litters H1 line L1 line 2nd 10 litters H2 line L2 line 3rd 10 litters C1 line 4th 10 litters C2 line Breeding & Selection Experiment The entire breeding experiment used a total of 14,184 mice

13. Differences within and between strains

14. Differences btw Selected Lines

15. Peculiar Association?

16. What Can We Do With Mouse Models

17. Database of Mouse Models

18. Disease Genes and Polymorphisms The Basics • Typically transgenic mice are used to show how the over-expression of a gene product affects physiology, behavior, etc. • Costs ≈ $3000 per mouse • Relatively straightforward procedure. • Transgenic mouse can be made in 3-6 months. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Methodology - Example - Issues Definitions Transgenesis: The transfer of human genes into animals or cultured cells, esp. to produce molecules with therapeutic value. Mutagenesis: The occurrence or induction of mutation

19. Disease Genes and Polymorphisms Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Methodology - Example - Issues Adapted from Hedari Laboratory, Wayne State Univ.

20. Disease Genes and Polymorphisms Alzheimer’s Transgenic Mice The APP transgenic mouse has been the most popularly used model with neuritic plaques conspicuously displayed by the age of 12-13 months of age Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Methodology - Example - Issues The development of transgenic mice that are doubly transgenic for APP and presenilin-1 (PS-1) yielded mice that developed plaques at a much earlier age than the singly transgenic APP mouse. Pictures of Alzheimer's Pathology Stain: Campbell-Switzer Silver Method Excert from: Neuroscience Associates Labs http://www.neuroscienceassociates.com/Stains/alzheimers.htm

21. Disease Genes and Polymorphisms Issues • Integration of transgene is random. • It may disrupt the function of another gene • May integrate into a part of the genome where gene expression is suppressed. • May integrate into a part of the genome under the control of a locus control region. • Number of copies cannot be controlled. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Methodology - Example - Issues Definitions Locus Control Region (LCR) – region of the chromosome that is responsible for the expression of linked genes to physiologically appropriate levels in specific tissues at developmentally appropriate time points.

22. Disease Genes and Polymorphisms Issues • Localization of expression cannot be controlled completely. • Over time, the transgene is frequently “shut off”. These issues can be overcome by generating several transgenic lines using the same construct and comparing the data across lines. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Methodology - Example - Issues

23. Disease Genes and Polymorphisms The Basics • Targeted mutant mice – Alteration of gene of interest through homologous recombination • The gene of interest can be eliminated (knock-out or null mutant) or altered (knock-in). • Loss of gene function or alteration of function is typically evaluated in KO and KI mice Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Methodology - Example - Issues Definitions Mutagenesis: The occurrence or induction of mutation

24. Disease Genes and Polymorphisms • The gene of interest can be “conditionally” deleted or altered. • Very labor intensive and technically challenging methods are involved. • Typical time to generate a null-mutant mouse is 1-3 years. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Methodology - Example - Issues

25. Disease Genes and Polymorphisms • Knock-out: Remove gene or exon of gene that renders the gene product non-functional. • Knock-in: Replace the natural gene or exon with an altered gene or exon that alters the function of the gene product. • Congenic*: Introgression of a locus of interest from another strain; selecting for a given marker in the donor strain. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression / Congenic* - Methodology - Example - Issues Definition Introgression: Infiltration of the genes of one species into the gene pool of another through repeated backcrossing of an interspecific hybrid with one of its parents.

26. Disease Genes and Polymorphisms • Conditional KO: Gene is modified so that it is deleted only under specific conditions. • Inducible/Tissue specific expression: Gene expression is regulated by a drug supplement (typically a chemical not endogenous to the mouse). Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues

27. Disease Genes and Polymorphisms Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues Definitiion Chimera - an animal that has two or more different populations of genetically distinct cells that originated in different zygotes Adapted from the Fred Hutchinson Cancer Research Center

28. Disease Genes and Polymorphisms Gene targeting in the mouse Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues

29. Disease Genes and Polymorphisms Problems with KO’s and KI’s • Targeting vector components (such as the marker used) may affect the expression of genes close to the mutated gene. • Potential developmental effects, such as: • Altered development • Compensatory mechanisms • Knock-out is lethal Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues

30. Disease Genes and Polymorphisms Problems with KO’s and KI’s • Genetic background: No behavioral trait is determined by a single gene. • It is expensive and labor intensive. • Mice take a long time to create. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues

31. Disease Genes and Polymorphisms Conditional targeted mutation: Use of Cre recombinase to delete genes in a temporal or spatial manner. • Cre recombinase is an enzyme that when in contact with LOXP sites flanking a region of DNA, it can alter the DNA between them. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues

32. Disease Genes and Polymorphisms Inducible Transgenic Mice • The inserted gene is activated at a desired developmental stage. • An novel transcription factor designed to bind to the gene and cause expression in the presence of an activator compound is used. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues

33. Disease Genes and Polymorphisms Problems of conditional KO’s and inducible transgenics • Limited availability of tissue-specific mice to knock-out or induce expression in a tissue specific fashion. • Conditional knockout may not delete the gene in all of the desired cells. • Gene induction/repression may not be efficiently regulated. Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues

34. Disease Genes and Polymorphisms NEIL 1 KO mouse model of Metabolic Syndrome (Vartanian et al, 2006) Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues Definitions Dyslipidemia – abnormal concentrations for lipoproteins in the blood. Deletion of the DNA glycosylase NEIL1 gene in mice results in severe obesity, fatty liver, dyslipidemia, and insulin resistance; symptoms that are consistent with metabolic syndrome in humans (a collective title for obesity, insulin resistance, dyslipidemia, and hypertension in humans)

35. Voluntary Ethanol Consumption in Chrna6 and Chrnb3 Knockout Mice (Vidable,. L, et al. 2008; Ehringer Lab) Types & Forms of nAch Receptors Human studies suggest that α6 & β3 nAch receptors are associated with alcohol. Alcohol modulates binding. Two Bottle Paradigm: Alcohol vs. Water

36. Voluntary Ethanol Consumption in Chrna6 and Chrnb3 Knockout Mice(Ehringer Lab, Summer ‘08) Chrna6 Knockouts Results Chrnb3 Knockouts Results Blue – Male; Green - Female

37. Disease Genes and Polymorphisms Examples of transgenic mice: Inducible Breast Cancer Inducible Memory Molecular defects of Alz. No Immune System Targeted Transgenesis in Mice - Methodology - Example - Issues Targeted Mutagenesis in Mice - Types of mutagenic mice - Knock-out/ Knock-in/ Conditional Knock-out/ Inducible expression - Methodology - Example - Issues

38. Alternate Resources