Construction of a knockout mouse model for combined methylmalonic aciduria and homocystinuria, cblC type (Mmachc) By Junhui Liu Department of Human Genetics McGill University, Montreal, Canada A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of Master © Junhui Liu, December 2009 1 TABLE OF CONTENTS ABSTRACT ..................................................................................................................... 5 RÉSUMÉ ......................................................................................................................... 6 ACKNOWLEDGEMENTS ............................................................................................ 7 LIST OF FIGURES ........................................................................................................ 8 LIST OF TABLES .......................................................................................................... 9 ABBREVIATIONS ....................................................................................................... 10 CHAPTER 1. Introduction ........................................................................................... 12 Chapter 1.1 Cobalamin ............................................................................................. 12 1.1.1 Vitamin B12 .................................................................................................. 12 1.1.2 Structure of cobalamin ................................................................................. 12 1.1.3 Distribution of cobalamins among life forms ............................................. 16 1.1.4 Cobalamin uptake and transport in mammals ........................................... 16 1.1.5 Intracellular cobalamin metabolism ........................................................... 18 1.1.6 Molecular and functional aspects of cobalamin metabolism ..................... 20 cblA, cblB and mut ............................................................................................. 21 cblA: ............................................................................................................... 22 cblB: ............................................................................................................... 23 mut: ................................................................................................................ 23 cblC, cblD and cblF ............................................................................................ 24 cblC: This will be discussed in details in Chapter 1.3 later. ....................... 24 cblD: ............................................................................................................... 24 2 cblF: ................................................................................................................ 24 cblE, cblG and cblD variant 1 ............................................................................ 25 cblE: ............................................................................................................... 26 cblG: ............................................................................................................... 26 Methylenetetrahydrofolate reductase (MTHFR) deficiency ........................... 27 1.1.7 Cobalamin and birth defects ........................................................................ 28 Chapter 1.2 Knockout mouse models for genes in cobalamin metabolism and transport pathway ..................................................................................................... 29 1.2.1 Methylenetetrahydrofolate reductase (Mthfr) knockout mouse model ..... 29 1.2.2 Methionine synthase (Mtr) knockout mouse model .................................... 30 1.2.3 Methionine synthase reductase (Mtrr) knockout mouse model ................. 30 1.2.4 Mut knockout mouse model ......................................................................... 31 1.2.5 Cubn knockout mouse model ....................................................................... 31 1.2.6 Amn mouse model ......................................................................................... 31 Chapter 1.3 MMACHC.............................................................................................. 32 RATIONALE AND OBJECTIVES OF RESEARCH ................................................ 38 CHAPTER 2. Materials and Methods ......................................................................... 39 Chapter 2.1 Generation of knockout mouse model: ............................................... 39 2.1.1 Gene trap information .................................................................................. 39 2.1.2 Evaluation of the gene trap construct ......................................................... 40 2.1.3 Generation of chimeric mice ........................................................................ 42 2.1.4 Generation of F1 mice and molecular characterization of the MMACHC knockout mice ........................................................................................................ 42 3 2.1.5 Embryo Studies ............................................................................................. 42 Chapter 2.2 Molecular Characterization ................................................................. 43 2.2.1 DNA extraction ............................................................................................. 43 2.2.2 RNA extraction ............................................................................................. 43 2.2.3 Sequencing analysis ...................................................................................... 44 Chapter 2.3 Biochemical analysis ............................................................................. 45 2.3.1 Measurement of metabolites in the blood and urine .................................. 45 2.3.2 Fibroblast studies ......................................................................................... 45 2.3.2a Cell culture .............................................................................................. 45 2.3.2b Cell lines .................................................................................................. 45 2.3.2c Propionate incorporation studies ........................................................... 46 2.3.2d Methyltetrahydrofolate incorporation studies ..................................... 47 CHAPTER 3. Results .................................................................................................... 48 Chapter 3.1 Molecular and biochemical characterization of adult mice ............... 48 Chapter 3.2 Molecular characterization of mouse embryos .................................. 55 Chapter 3.3 Embryo morphology ............................................................................ 56 Chapter 3.4 MCM and MS function of mouse fibroblasts ..................................... 60 CHAPTER 4. Discussion .............................................................................................. 65 SUMMARY AND CONCLUSION .............................................................................. 73 CLAIMS OF ORIGINALITY ...................................................................................... 74 BIBLIOGRAPHY ......................................................................................................... 75 APPENDIX A: PUBLICATIONS AND PRESENTATIONS .................................... 85 APPENDIX B: ETHICS AND CERTIFICATES ....................................................... 90 4 ABSTRACT The MMACHC gene is responsible for cblC, the most common inborn error of cobalamin metabolism in man. We created a knockout mouse model for its ortholog, Mmachc. Embryonic stem cells from 129 strain mice heterozygous for Mmachc containing a gene trap in intron 1 were injected into blastocysts from c57B6 mice to generate chimeras. Crossing chimeric males to c57B6 females generated heterozygous F1 mice. Geonotyping of F2 mice showed 36 wild type, 71 heterozygous and 3 homozygous. The three homozygous mutant F2 mice had a normal phenotype possibly due to alternative splicing causing expression of wild type Mmachc. Genotyping of embryos showed absence of homozygous mutants at e17.5, suggesting they died earlier. We observed phenotypes including open neural folds, amnionless-like, holoprosencephaly and abnormal limbs and face in individual homozygous mutant embryos. This work enables us to further clarify functions of Mmachc and the role of it in birth defects. 5 RÉSUMÉ Chez l’homme, le gène MMACHC est responsable de la maladie cblC qui est l’erreur innée la plus commune de sentier de métabolique de la cobalamine. Nous avons créé un modèle de souris knockout pour son orthologue--Mmachc. Des cellules souches embryonnaires de type 129 heterozygotes pour un gène piégé dans l’intron 1 de Mmachc ont été injectées dans des blastocystes de type c57B6 afin de produire des chimères. Le croisement de mâles chimériques et de femelles de type c57B6 a produit des mutants hétérozygotes F1. Le génotypage a démontré 36 souris de type sauvage, 71 hétérozygotes et 3 homozygotes. Le génotypage a démontré l'absence d'embryons homozygotes mutants à e17.5, suggérant que les embryons homozygotes mutants sont morts avant e17.5. Nous avons observé des dysmorphologies telles que des plis neuronaux ouverts, un phenotype similaire à ‘amnionless’, de l’holoprosencephaly et des membres et visages anormaux dans les embryons homozygotes mutants. Les trois souris homozygotes