Introduction to Mouse genetic model second
- 1. Mouse Genetic Models II Mouse Genetic Models II M. Sc. 2006
- 2. Hypothesis-driven Expensive failures phenotype Capn5 Capn5LacZ /+ +/+ Disadvantages of Reverse Genetics Disadvantages of Reverse Genetics
- 3. Systematic approaches Systematic approaches phenotype phenotype gene gene Forward genetics (study disease biology) Forward genetics (study disease biology) Reverse genetics (study existing (target) genes) Reverse genetics (study existing (target) genes) +/+ m/ m m/+ m/+ +/+ m/ m m/+ m/+
- 4. Systematic approaches Systematic approaches phenotype gene Forward genetics (study disease biology) Forward genetics (study disease biology) +/+ m/ m m/+ m/+ • Spontaneous mutations • ENU-induced mutations
- 5. Systematic approaches Systematic approaches phenotype gene Forward genetics (study disease biology) Forward genetics (study disease biology) +/+ m/ m m/+ m/+ • Spontaneous mutations • ENU-induced mutations
- 6. ob and db mice ob and db mice ob/ob mice •ob = obese •Discovered in 1950 in Jackson Laboratories •recessive genetic obesity that results in sterile adult mice with over 50% fat db/db mice •db = diabetes •Discovered in 1966 •Obese •Excess water intake and urination •Also hyperglycaemic
- 7. Obesity Obesity • a major health issue in much of the human population • estimated that over 30% of the population is overweight by at least 20% • at least $30 billion are spent to treat obesity in the US annually • most attempts to treat obesity to date, except for the several types of surgical removal of the tissue, have failed to result in a sustained reduction of obesity
- 8. Parabiotic experiments Parabiotic experiments Douglas Coleman, 1973 • the ob mouse lost weight and died • normal mouse had no food in its stomach and low blood sugar levels, typical of starvation • suggesting that the ob factor was produced by the db mouse and circulates • the obese mouse lost weight but did not die • suggesting a circulating ob factor • the normal mouse lost weight and died after 3 weeks • normal mouse had no food in its stomach and low blood sugar levels, typical of starvation • suggesting that the db component does not circulate, and the ob factor is overexpressed by the db mouse)
- 9. Parabiotic experiments Parabiotic experiments Douglas Coleman, 1973 ob ob ob ob ob ob ob X db/db mice make the circulating factor but their brains cannot respond to it to reduce food intake ob/ob mice fail to make a circulating factor but their brains can still respond to it to reduce food intake X
- 10. Identification of the Identification of the ob ob mutation mutation Jeffrey Friedman’s group, 1994 • End of an 8-year-long search • Specific gene defect in ob mice led to lack of a circulating factor named leptin • Patent for leptin sold for an up-front US$ 20 million to Amgen in 1995 (astonishing amount at the time) • db mice had mutation in the gene for the receptor for leptin
- 11. Toll receptors Toll receptors Critical proteins linking innate and adaptive immunity QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. C3HeB/HeJ - insensitive to LPS QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
- 12. Random integration of transgenes Random integration of transgenes •Generally a disadvantage as leads to inconsistency between transgenic lines e.g. if integrates close to a regulatory element •If inserts within a gene, can inactivate the gene e.g. Invs mutant tyrosinase Deleted region 40 kb one transgenic line demonstrated situs inversus inv/inv +/+
- 13. Systematic approaches Systematic approaches phenotype gene Forward genetics (study disease biology) Forward genetics (study disease biology) +/+ m/ m m/+ m/+ • Spontaneous mutations • ENU-induced mutations
- 14. N N-ethyl- -ethyl-N N-nitrosourea (ENU) -nitrosourea (ENU) (alkylating agent O6 guanine, 04 thymine, O6 adenine) 44% A:T to T:A 38% A:T to G:C 8% G:C to A:T 3% G:C to C:G 5% A:T to C:G 2% G:C to T:A 64% missense mutations 10% nonsense mutations 26% splicing errors Functional mutation in a particular gene = 1 in 700 O N C NH2 O H3C CH2
- 15. ENU Mutagenesis ENU Mutagenesis X ENU G0 X m + + + X m + m + m + m + m + + m G1 G2 G3 Screen for dominant phenotypes Screen for recessive phenotypes
- 16. Why look for dominant Why look for dominant mutations? mutations? Estimated that: • 49% of inherited diseases in humans are recessive but many of these are isolated cases. Majority of patients with a genetic disease are dominant
- 17. Sensory Sensory Hearing, vision Neurological/behavioural Neurological/behavioural Circadian/anxiety, myopathies Hormonal/metabolic Hormonal/metabolic Type II diabetes Core Screens Core Screens Immunology Immunology Innate immunity, T-cell Developmental Developmental Gonad, left-right asymmetry, spleen X-ray X-ray Kidney stones, scoliosis Growth, Ageing Growth, Ageing Dysmorphology, misc. Dysmorphology, misc.
- 18. Classes of informative ENU Classes of informative ENU mutants mutants •New mouse models/novel phenotypes •Novel phenotypes in genes of “known” function •Phenotypes in genes of unknown function
- 19. Classes of informative ENU Classes of informative ENU mutants mutants •New mouse models/novel phenotypes •Novel phenotypes in genes of “known” function •Phenotypes in genes of unknown function
- 20. New mouse models/novel phenotypes New mouse models/novel phenotypes (MRC Harwell) Belted Short forelimb Walking backwards Circling Activity
- 21. Dilated Cardiomyopathy Dilated Cardiomyopathy Age = 10 weeks; weight = 70g Skin, control, x40 Skin, Chf/+, x40 •Atrial thrombosis and subcutaneous oedema •Maps to chromosome 16 Atrial thrombosis, Chf
- 22. Renal calcification Renal calcification Rajesh Thakker et al., Univ. of Oxford
- 23. Classes of informative ENU Classes of informative ENU mutants mutants •New mouse models/novel phenotypes •Novel phenotypes in genes of “known” function •Phenotypes in genes of unknown function
- 24. Novel phenotype in gene of known Novel phenotype in gene of known function function Loa, Cra1 - A new model for motor neuron degeneration Fatal degeneration of motor neurons Amyotrophic Lateral Sclerosis, Spinal Muscular Atrophy Progressive and specific process >95% of ALS of unknown aetiology WT Het.
- 25. Loa and Cra1: Loa and Cra1: progressive motor neuron degeneration progressive motor neuron degeneration Progressive loss of grip strength Progressive loss of spinal cord anterior horn a motor neurons and altered muscle fibre morphology
- 26. ....Caused by Subtle Mutation in Dynein ....Caused by Subtle Mutation in Dynein Heavy Chain 1 gene Heavy Chain 1 gene Loa (binding site for intermediate chains) Cra1 (heavy chain homodimerization domain)
- 27. Specific Impairment of Specific Impairment of Retrograde Axonal Transport Retrograde Axonal Transport Hafezparast et al. (2003) Science 300, 808 Retrograde transport (positive) is impaired in Loa/Loa mice Marked reduction in high speed carriers in axonal retrograde transport
- 28. Loss of Loss of Dnhc1 Dnhc1 function is cell lethal function is cell lethal Dnchc1 KO Harada et al., JCB 1998 wt mut
- 29. Classes of informative ENU Classes of informative ENU mutants mutants •New mouse models/novel phenotypes •Novel phenotypes in genes of “known” function •Phenotypes in genes of unknown function
- 30. Wild type Mut Wild type Mut
- 31. a W T M u t Males W T M u t Females ** * ** ** * ** b c d Mut male Wild type male Mut female Wild type female Mut Mut Wild type c d
- 32. E->K an domains possible an domains serine-rich domain C-terminal domain nuclear localisation signal
- 33. Different types of mutations Different types of mutations • Nullimorph • Hypomorph • Hypermorph • Neomorph • Antimorph Loss-of-function Gain-of-function
- 34. Antimorph - Dominant Antimorph - Dominant negative negative QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. RAR-RXR heterodimer
- 35. Dominant negative Dominant negative (p53) Zhang, Z. et al. Cancer Res 2002;62:3024-3029 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 135A->V wt/wt T135V
- 36. p53 dominant negative mechanism p53 dominant negative mechanism of action of action QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Oligomerization of p53 is required for in vivo p53 activity and contributes to the dominant- negative effect of some p53 mutants. The core domain of p53 in complex with DNA bearing the consensus p53-binding site is shown here. DNA- binding by p53 is essential to its activity as a transcriptional activator of the Cdk inhibitor p21 (Cip1).
- 37. Hypermorph Hypermorph severe inflammation of the paw severe arthritis 50 100 150 200 0.21 0.23 0.25 0.27 0.29 fluorescence Time (secs) after stimulation of FcgR in macrophages Ali5 wt
- 38. Neomorph Neomorph e.g. spontaneous mutation Mc1R Mc2R Mc3R Mc4R Mc5R -MSH -MSH ACTH melanocytes adrenal adipose brain pancreas placenta brain muscle adipose tisse ubiquitous agouti
- 39. Forward and reverse genetics are Forward and reverse genetics are synergistic synergistic yellow obese mouse Mc4R Mc5R melanocortin system Msh receptors 1-5 Forward genetics Forward genetics Reverse genetics Reverse genetics Merck L’Oreal Gene-driven mutagenesis Positional cloning
- 40. Conclusion Conclusion • Forward and reverse genetic strategies are synergistic • Both are valuable to elucidate genetic pathways • Large-scale projects using both strategies have been implemented • One major goal is to define the function of all genes in the mammalian genome
- 41. Any Questions?
Editor's Notes
- #8: The db mouse is producing some “satiety factor” but is unable to respond to it i.e. the db mouse lacks a molecular receptor for the “satiety factor”
- #9: In fact, db mice have 20-fold normal levels of the circulating factor presumably due to some feedback loop
- #12: Invs = inversin Note that rhs embryo is not actually a wild type embryo but is an inv/inv embryo rescued with a transgene Answer to question: Unexoected? (only a hint to look further) 2. Outside domain of transgene expression or lineages that expressed transgene 2. Only a single transgenic line gives the phenotype
- #16: Why??????
- #34: The MMTV-LTR has been widely used to target transgene expression to the mammary epithelium and was chosen in this study because of our particular interest in breast cancer. Retinoic acid suppresses cell growth and promotes cell differentiation, and pharmacological retinoic acid receptor (RAR) activation is anti-tumorigenic. This begs the question of whether chronic physiological RAR activation by endogenous retinoids is likewise anti-tumorigenic. RARα bearing a G303E point mutation in the ligand binding domain (RARαG303E) binds ligand poorly but retains the ability to heterodimerize with RXR; when transfected into RA-sensitive cells, RARαG303E inhibited the transcriptional activity of endogenous RAR by nearly 100% at physiological RA concentrations (1ﾐ10 nM)
- #35: histologically most of the uterine sarcomas were leiomyosarcoma, which was composed of the tumor cells with large nuclei and associated with abnormal mitosis (>50 mitosis cell/50 high-power fields)
- #36: The tetramerization domain of p53 appears here. Oligomerization of p53 is required for in vivo p53 activity and contributes to the dominant-negative effect of some p53 mutants.
- #38: And a really nice example of this started with the discovery of the yellow obese mouse, a spontaneous mouse mutant with a yellow coat colour and obesity, type 2 diabetes, increased somatic growth and tumourigenesis. The common, unifying molecular feature of all of the yellow obese agouti mutations is the ubiquitous and strong expression of the wild-type agouti coding sequences from another transcriptional promoter due to a 170kb deletion of most of the coding region of an upstream gene called Raly, placing the agouti coding exons under the transcriptional control of the Raly promoter, which leads to the the ubiquitous expression of the agouti protein. The yellow coat colour is the result of agouti chronically antagonizing the binding of alpha-melanocyte stimulating hormone (-MSH) to the melanocortin 1 receptor (Mc1r), switching melanin synthesis from eumelanin (black/brown) to phaeomelanin (red/yellow). The melanocortin system was therefore implicated in the disease processes observed in this mutant, and these receptors were then analyzed. Infact, the obese phenotype results from agouti protein antagonizing the binding of a-MSH to Mc3r and/or Mc4r. Ubiquitous expression results in chromic antagonism of Mc4R in the hypothalamus (increased feeding) and Mc3R (more efficient storage of calories) ACTH -
- #39: Now these 2 approaches, as will become evident from this seminar, are synergistic. And a really nice example of this started with the discovery of the yellow obese mouse, a spontaneous mouse mutant with a yellow coat colour and obesity, type 2 diabetes, increased somatic growth and tumourigenesis. The common, unifying molecular feature of all of the yellow obese agouti mutations is the ubiquitous and strong expression of the wild-type agouti coding sequences from another transcriptional promoter due to a 170kb deletion of most of the coding region of an upstream gene called Raly, placing the agouti coding exons under the transcriptional control of the Raly promoter, which leads to the the ubiquitous expression of the agouti protein. The yellow coat colour is the result of agouti chronically antagonizing the binding of alpha-melanocyte stimulating hormone (-MSH) to the melanocortin 1 receptor (Mc1r), switching melanin synthesis from eumelanin (black/brown) to phaeomelanin (red/yellow). The melanocortin system was therefore implicated in the disease processes observed in this mutant, and these receptors were then analyzed. Infact, the obese phenotype results from agouti protein antagonizing the binding of a-MSH to Mc3r and/or Mc4r. Chen et al. (1997) focussed on Mc5R and when this was knocked out, there was no obese phenotype. They then put the mice through a series of tests. One of these is the stress-induced analgaesia test. The emotional responses induced when individuals are confronted with noxious or threatening stimuli is accompanied by an analgesic effect that might be elicited by activation of intrinsic pain-inhibitory systems, one of the basic adaptive mechanisms of animals and humans. The stress was induced by allowing the mice to swim in a water bath for a short period. An alert individual noted that the knockout mice took much longer to dry than the controls and further investigation revealed a severe defect in water repulsion and thermoregulation in the Mc5R-null due to decreased production of sebaceous lipids. So whereas Mc4R is being investigated by Merck as a potential target for obesity/diabetes, Mc5R is being studied by L’Oreal.