Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67206Full metadata record
| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳佑宗 | |
| dc.contributor.author | Shin-Yu Wang | en |
| dc.contributor.author | 王昕妤 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:23:31Z | - |
| dc.date.available | 2017-09-12 | |
| dc.date.copyright | 2017-09-12 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-09 | |
| dc.identifier.citation | 1. Lindahl, T. and D.E. Barnes, Repair of endogenous DNA damage. Cold Spring Harb Symp Quant Biol, 2000. 65: p. 127-33.
2. Hanahan, D. and R.A. Weinberg, Hallmarks of cancer: the next generation. Cell, 2011. 144(5): p. 646-74. 3. Jackson, S.P. and J. Bartek, The DNA-damage response in human biology and disease. Nature, 2009. 461(7267): p. 1071-8. 4. Li, G.M., Mechanisms and functions of DNA mismatch repair. Cell Res, 2008. 18(1): p. 85-98. 5. Kunkel, T.A. and D.A. Erie, DNA mismatch repair. Annu Rev Biochem, 2005. 74: p. 681-710. 6. Jiricny, J., The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol, 2006. 7(5): p. 335-46. 7. Hegde, M.L., T.K. Hazra, and S. Mitra, Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells. Cell Res, 2008. 18(1): p. 27-47. 8. David, S.S., V.L. O'Shea, and S. Kundu, Base-excision repair of oxidative DNA damage. Nature, 2007. 447(7147): p. 941-50. 9. Shuck, S.C., E.A. Short, and J.J. Turchi, Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology. Cell Res, 2008. 18(1): p. 64-72. 10. Fousteri, M. and L.H. Mullenders, Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects. Cell Res, 2008. 18(1): p. 73-84. 11. Li, X. and W.D. Heyer, Homologous recombination in DNA repair and DNA damage tolerance. Cell Res, 2008. 18(1): p. 99-113. 12. Sung, P. and H. Klein, Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol, 2006. 7(10): p. 739-50. 13. Moynahan, M.E. and M. Jasin, Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat Rev Mol Cell Biol, 2010. 11(3): p. 196-207. 14. Weterings, E. and D.J. Chen, The endless tale of non-homologous end-joining. Cell Res, 2008. 18(1): p. 114-24. 15. Chiruvella, K.K., Z. Liang, and T.E. Wilson, Repair of double-strand breaks by end joining. Cold Spring Harb Perspect Biol, 2013. 5(5): p. a012757. 16. Lieber, M.R., The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem, 2010. 79: p. 181-211. 17. van Gent, D.C. and M. van der Burg, Non-homologous end-joining, a sticky affair. Oncogene, 2007. 26(56): p. 7731-40. 18. Gerson, S.L., MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer, 2004. 4(4): p. 296-307. 19. Pegg, A.E., Repair of O(6)-alkylguanine by alkyltransferases. Mutat Res, 2000. 462(2-3): p. 83-100. 20. Weller, M., et al., MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol, 2010. 6(1): p. 39-51. 21. Lord, C.J. and A. Ashworth, The DNA damage response and cancer therapy. Nature, 2012. 481(7381): p. 287-94. 22. Loeb, L.A., K.R. Loeb, and J.P. Anderson, Multiple mutations and cancer. Proc Natl Acad Sci U S A, 2003. 100(3): p. 776-81. 23. Cancer Genome Atlas, N., Comprehensive molecular portraits of human breast tumours. Nature, 2012. 490(7418): p. 61-70. 24. Cancer Genome Atlas Research, N., Integrated genomic analyses of ovarian carcinoma. Nature, 2011. 474(7353): p. 609-15. 25. Puente, X.S., et al., Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature, 2011. 475(7354): p. 101-5. 26. Quesada, V., et al., Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. Nat Genet, 2011. 44(1): p. 47-52. 27. Cancer Genome Atlas, N., Comprehensive molecular characterization of human colon and rectal cancer. Nature, 2012. 487(7407): p. 330-7. 28. Palombo, F., et al., Mismatch repair and cancer. Nature, 1994. 367(6462): p. 417. 29. Bronner, C.E., et al., Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature, 1994. 368(6468): p. 258-61. 30. Grasso, C.S., et al., The mutational landscape of lethal castration-resistant prostate cancer. Nature, 2012. 487(7406): p. 239-43. 31. Fishel, R. and T. Wilson, MutS homologs in mammalian cells. Curr Opin Genet Dev, 1997. 7(1): p. 105-13. 32. Kolodner, R., Biochemistry and genetics of eukaryotic mismatch repair. Genes Dev, 1996. 10(12): p. 1433-42. 33. Warren, J.J., et al., Structure of the human MutSalpha DNA lesion recognition complex. Mol Cell, 2007. 26(4): p. 579-92. 34. Surtees, J.A. and E. Alani, Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination. J Mol Biol, 2006. 360(3): p. 523-36. 35. Pluciennik, A., et al., PCNA function in the activation and strand direction of MutLalpha endonuclease in mismatch repair. Proc Natl Acad Sci U S A, 2010. 107(37): p. 16066-71. 36. Kadyrov, F.A., et al., Endonucleolytic function of MutLalpha in human mismatch repair. Cell, 2006. 126(2): p. 297-308. 37. Buermeyer, A.B., et al., Mammalian DNA mismatch repair. Annu Rev Genet, 1999. 33: p. 533-64. 38. Poulogiannis, G., I.M. Frayling, and M.J. Arends, DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome. Histopathology, 2010. 56(2): p. 167-79. 39. Lengauer, C., K.W. Kinzler, and B. Vogelstein, Genetic instability in colorectal cancers. Nature, 1997. 386(6625): p. 623-7. 40. Kim, T.M., P.W. Laird, and P.J. Park, The landscape of microsatellite instability in colorectal and endometrial cancer genomes. Cell, 2013. 155(4): p. 858-68. 41. Leach, F.S., et al., Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell, 1993. 75(6): p. 1215-25. 42. Parsons, R., et al., Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell, 1993. 75(6): p. 1227-36. 43. Haggar, F.A. and R.P. Boushey, Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors. Clin Colon Rectal Surg, 2009. 22(4): p. 191-7. 44. Balmana, J., et al., Prediction of MLH1 and MSH2 mutations in Lynch syndrome. JAMA, 2006. 296(12): p. 1469-78. 45. Sinicrope, F.A. and Z.J. Yang, Prognostic and predictive impact of DNA mismatch repair in the management of colorectal cancer. Future Oncol, 2011. 7(3): p. 467-74. 46. Nagasaka, T., et al., Somatic hypermethylation of MSH2 is a frequent event in Lynch Syndrome colorectal cancers. Cancer Res, 2010. 70(8): p. 3098-108. 47. Bertagnolli, M.M., et al., Microsatellite instability and loss of heterozygosity at chromosomal location 18q: prospective evaluation of biomarkers for stages II and III colon cancer--a study of CALGB 9581 and 89803. J Clin Oncol, 2011. 29(23): p. 3153-62. 48. Ellison, A.R., J. Lofing, and G.A. Bitter, Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain. Nucleic Acids Res, 2004. 32(18): p. 5321-38. 49. Herman, J.G., et al., Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A, 1998. 95(12): p. 6870-5. 50. Toyota, M., et al., CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci U S A, 1999. 96(15): p. 8681-6. 51. Lanza, G., et al., Immunohistochemical pattern of MLH1/MSH2 expression is related to clinical and pathological features in colorectal adenocarcinomas with microsatellite instability. Mod Pathol, 2002. 15(7): p. 741-9. 52. Lanza, G., et al., Immunohistochemical test for MLH1 and MSH2 expression predicts clinical outcome in stage II and III colorectal cancer patients. J Clin Oncol, 2006. 24(15): p. 2359-67. 53. Tokairin, Y., et al., Accelerated growth of intestinal tumours after radiation exposure in Mlh1-knockout mice: evaluation of the late effect of radiation on a mouse model of HNPCC. Int J Exp Pathol, 2006. 87(2): p. 89-99. 54. Kawate, H., et al., A defect in a single allele of the Mlh1 gene causes dissociation of the killing and tumorigenic actions of an alkylating carcinogen in methyltransferase-deficient mice. Carcinogenesis, 2000. 21(2): p. 301-5. 55. Hegan, D.C., et al., Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6. Carcinogenesis, 2006. 27(12): p. 2402-8. 56. Reitmair, A.H., et al., MSH2 deficient mice are viable and susceptible to lymphoid tumours. Nat Genet, 1995. 11(1): p. 64-70. 57. de Wind, N., et al., Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer. Cell, 1995. 82(2): p. 321-30. 58. Prolla, T.A., et al., Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair. Nat Genet, 1998. 18(3): p. 276-9. 59. Edelmann, W., et al., Tumorigenesis in Mlh1 and Mlh1/Apc1638N mutant mice. Cancer Res, 1999. 59(6): p. 1301-7. 60. Egashira, A., et al., Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes. DNA Repair (Amst), 2002. 1(11): p. 881-93. 61. Kawate, H., et al., Separation of killing and tumorigenic effects of an alkylating agent in mice defective in two of the DNA repair genes. Proc Natl Acad Sci U S A, 1998. 95(9): p. 5116-20. 62. Kucherlapati, M.H., et al., An Msh2 conditional knockout mouse for studying intestinal cancer and testing anticancer agents. Gastroenterology, 2010. 138(3): p. 993-1002 e1. 63. Wojciechowicz, K., et al., Temozolomide increases the number of mismatch repair-deficient intestinal crypts and accelerates tumorigenesis in a mouse model of Lynch syndrome. Gastroenterology, 2014. 147(5): p. 1064-72 e5. 64. Kucherlapati, M.H., et al., Genotype directed therapy in murine mismatch repair deficient tumors. PLoS One, 2013. 8(7): p. e68817. 65. Reiss, C., et al., Conditional inactivation of MLH1 in thymic and naive T-cells in mice leads to a limited incidence of lymphoblastic T-cell lymphomas. Leuk Lymphoma, 2010. 51(10): p. 1875-86. 66. Petters, R.M. and J.R. Sommer, Transgenic animals as models for human disease. Transgenic Res, 2000. 9(4-5): p. 347-51; discussion 345-6. 67. Walrath, J.C., et al., Genetically engineered mouse models in cancer research. Adv Cancer Res, 2010. 106: p. 113-64. 68. Russell, W.L., X-ray-induced mutations in mice. Cold Spring Harb Symp Quant Biol, 1951. 16: p. 327-36. 69. Russell, L.B., et al., Chlorambucil effectively induces deletion mutations in mouse germ cells. Proc Natl Acad Sci U S A, 1989. 86(10): p. 3704-8. 70. Yu, Y. and A. Bradley, Engineering chromosomal rearrangements in mice. Nat Rev Genet, 2001. 2(10): p. 780-90. 71. Ramirez-Solis, R., P. Liu, and A. Bradley, Chromosome engineering in mice. Nature, 1995. 378(6558): p. 720-4. 72. Zheng, B., et al., Engineering mouse chromosomes with Cre-loxP: range, efficiency, and somatic applications. Mol Cell Biol, 2000. 20(2): p. 648-55. 73. Buchholz, F., et al., Inducible chromosomal translocation of AML1 and ETO genes through Cre/loxP-mediated recombination in the mouse. EMBO Rep, 2000. 1(2): p. 133-9. 74. Collins, E.C., et al., Inter-chromosomal recombination of Mll and Af9 genes mediated by cre-loxP in mouse development. EMBO Rep, 2000. 1(2): p. 127-32. 75. Merscher, S., et al., TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell, 2001. 104(4): p. 619-29. 76. Lindsay, E.A., et al., Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice. Nature, 2001. 410(6824): p. 97-101. 77. Gaj, T., C.A. Gersbach, and C.F. Barbas, 3rd, ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol, 2013. 31(7): p. 397-405. 78. Urnov, F.D., et al., Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature, 2005. 435(7042): p. 646-51. 79. Li, H., et al., In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature, 2011. 475(7355): p. 217-21. 80. Gaj, T., et al., Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods, 2012. 9(8): p. 805-7. 81. Bedell, V.M., et al., In vivo genome editing using a high-efficiency TALEN system. Nature, 2012. 491(7422): p. 114-8. 82. Wiedenheft, B., S.H. Sternberg, and J.A. Doudna, RNA-guided genetic silencing systems in bacteria and archaea. Nature, 2012. 482(7385): p. 331-8. 83. Sternberg, S.H., et al., DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 2014. 507(7490): p. 62-7. 84. Maddalo, D., et al., In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature, 2014. 516(7531): p. 423-7. 85. Ran, F.A., et al., Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 2013. 8(11): p. 2281-308. 86. Deveau, H., J.E. Garneau, and S. Moineau, CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol, 2010. 64: p. 475-93. 87. Horvath, P. and R. Barrangou, CRISPR/Cas, the immune system of bacteria and archaea. Science, 2010. 327(5962): p. 167-70. 88. Garneau, J.E., et al., The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010. 468(7320): p. 67-71. 89. Anders, C., et al., Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 2014. 513(7519): p. 569-73. 90. Zlotorynski, E., Genome engineering: NHEJ and CRISPR-Cas9 improve gene therapy. Nat Rev Mol Cell Biol, 2016. 18(1): p. 4. 91. Su, T., et al., A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome. Sci Rep, 2016. 6: p. 37895. 92. He, X., et al., Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res, 2016. 44(9): p. e85. 93. Wang, B., et al., Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes. Biotechniques, 2015. 59(4): p. 201-2, 204, 206-8. 94. Mukherjee, S. and M. Feig, Conformational change in MSH2-MSH6 upon binding DNA coupled to ATPase activity. Biophys J, 2009. 96(11): p. L63-5. 95. Xue, W., et al., CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature, 2014. 514(7522): p. 380-4. 96. Olmsted, J.B., et al., Isolation of microtubule protein from cultured mouse neuroblastoma cells. Proc Natl Acad Sci U S A, 1970. 65(1): p. 129-36. 97. Chahwan, R., et al., The ATPase activity of MLH1 is required to orchestrate DNA double-strand breaks and end processing during class switch recombination. J Exp Med, 2012. 209(4): p. 671-8. 98. Yan, T., et al., DNA mismatch repair (MMR) mediates 6-thioguanine genotoxicity by introducing single-strand breaks to signal a G2-M arrest in MMR-proficient RKO cells. Clin Cancer Res, 2003. 9(6): p. 2327-34. 99. Karran, P., Thiopurines, DNA damage, DNA repair and therapy-related cancer. Br Med Bull, 2006. 79-80: p. 153-70. 100. Kucera, G.T., D.M. Bortner, and M.P. Rosenberg, Overexpression of an Agouti cDNA in the skin of transgenic mice recapitulates dominant coat color phenotypes of spontaneous mutants. Dev Biol, 1996. 173(1): p. 162-73. 101. Toft, N.J., et al., Heterozygosity for p53 promotes microsatellite instability and tumorigenesis on a Msh2 deficient background. Oncogene, 2002. 21(41): p. 6299-306. 102. Lee, K., E. Tosti, and W. Edelmann, Mouse models of DNA mismatch repair in cancer research. DNA Repair (Amst), 2016. 38: p. 140-6. 103. Harvey, M., et al., Genetic background alters the spectrum of tumors that develop in p53-deficient mice. FASEB J, 1993. 7(10): p. 938-43. 104. Hayashi, S., T. Tenzen, and A.P. McMahon, Maternal inheritance of Cre activity in a Sox2Cre deleter strain. Genesis, 2003. 37(2): p. 51-3. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67206 | - |
| dc.description.abstract | 癌症為國人十大死因之首，癌症基因體也是學術研究的重點之一。在細胞中，一般的代謝活動和環境因素都能造成DNA損傷，這些損傷有可能造成DNA的破壞，改變基因編碼的內容，或是引發在細胞基因體中的潛在有害突變，進而造成基因體不穩定性，大幅增加細胞癌化的可能。因此當細胞產生DNA損傷時，DNA就會啟動自我修復機制。DNA 修復能力對生物體維持基因體的完整性是至關重要的。我們想要針對抑癌基因(tumor suppressor genes)中的DNA修復基因(DNA repair genes) 進行相關研究。Mlh1, Msh2 這兩個基因，是參與DNA 錯配修復(mismatch repair) 最重要的兩個角色，其主要功能在修復DNA複製過程中所產生的鹼基-鹼基錯配。我們想藉由近年來興起CRISPR/Cas9 的基因體編輯技術，在小鼠活體內進行Mlh1, Msh2的破壞來研究其分子間之遺傳交互作用與癌症進程的相關性。CRISPR/Cas9的基因體編輯技術，可以讓我們在不必經由冗長、費力的動物交配、繁殖之下，於活體內同時針對單個或多個基因製造突變的體細胞群。我們想建立一個攜帶著可藉由組織特異性環化重組酶(Cre)啟動的iCas9、帶有毛色標幟的、並針對Mlh1,Msh2進行破壞的sgRNA的複合轉基因sgRNA-iCas9小鼠模式，稱為ICE CRIM(Inducible Cas9 Effector/ CRIspr Mutagen)。這個小鼠模式將可以搭配不同組織特異性重組酶(Cre)小鼠，進而條件式的啟動Cas9的活化。在小鼠體內特定組織進行Mlh1,Msh2的破壞，提供一個Mlh1,Msh2突變異質性高、易發癌症的背景，未來可能讓我們得以在突變的體細胞群上進行正向遺傳篩選或檢驗不同癌症基因突變事件間對於癌症起始、進程的合作關係。 | zh_TW |
| dc.description.abstract | Cancer is the leading cause of mortality worldwide. Cancer genomics is a hot topic for biomedical research. The process a cell changes from a normal somatic cell into a cancer cell involves many factors. Genome instability and the accumulation of mutations are highly correlated to the incidence of cancer. Many studies found that oncogenes and tumor suppressor genes are directly related to cancer. Here I want to focus on the DNA repair genes, a small group of tumor suppressor genes. Cells in
human body receive tens of thousands of DNA lesions per day. When a cell suffered from DNA damage, the cell will start self-repair mechanisms, so that most of the mutations can be restored. DNA repair pathways can therefore reduce the mutation rate and safeguard the integrity of genome. One of the DNA repair mechanisms is DNA mismatch repair (MMR). The MMR machinery recognize and repair base-base mismatches produced during DNA replication. Mlh1 and Msh2 are two major players in DNA mismatch repair. Here I would like to generate a mouse model deficient in DNA mismatch repair by destroying Mlh1 and Msh2 simultaneously. I used the latest CRISPR/Cas9 gene editing technology to perform somatic mutagenesis of target gene(s) in vivo. We can get single or multiple genes mutated with minimum animal breeding efforts. It is also possible to investigate the relationship between genes and human diseases by manipulating gene editing in animals. I would like to generate a coat-color iii tagged transgenic mouse carrying an inducible Cas9 effector (ICE) cassette, which is activable by Cre, and CRIspr Mutagens (CRIM), the sgRNAs target either Mlh1 or Msh2. Mating with Cre transgenic mouse, Cas9 can be conditionally activated, and Mlh1 and Msh2 will be destroyed in different tissues in an ICE CRIM mouse. In the future, our model could potentially provide a ‘mutator phenotype’ background caused by Mlh1 and Msh2 heterogeneous mutations, allowing us to discover the genetic interactions between different cancer gene mutation events for cancer initiation and evolution in the mutant somatic cells. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:23:31Z (GMT). No. of bitstreams: 1 ntu-106-R04455001-1.pdf: 6813360 bytes, checksum: e25d8f1eddfeb3cb6c294907456a34a7 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 中文摘要…………………………………………………………………………………i
Abstract………………………………………………………………………………….ii Contents…………………………………………………………………………………iv List of figures………………………………………………………………………vi List of tables…………………………………………………………………………ix List of supplementary information……………………………………………………x 1. Introduction…………………………………………………………………………1 1.1 DNA repair pathways defects and genomic instability in cancer……………1 1.2 MLH1 and MSH2 are the major players in DNA mismatch repair (MMR)……………………………………………………………2 1.3 DNA mismatch repair (MMR) deficient mouse models for human cancer research………………………………………………………………………3 1.4 Genetically engineered mouse model for research…………………………4 1.5 The CRISPR/Cas9 System…………………………………………………5 2. Materials and methods………………………………………………………………8 2.1 spCas9 plasmids………………………………………………………………8 2.2 Cell culture……………………………………………………………………8 2.3 Plasmid transfection…………………………………………………………9 2.4 Surveyor nuclease assay……………………………………………………10 2.5 Protein extraction and Western blotting………………………………11 2.6 Cell viability assay…………………………………………………………12 2.7 ColonyAssay………………………………………………………………13 2.8 Construct DNA preparation for pronucleus microinjection…………………14 2.9 Mouse genotyping…………………………………………………………15 2.10 Tissue RNA isolation………………………………………………………15 2.11 Retrotranscription and RT-qPCR……………………………………………16 2.12 Tissue DNA extraction and sequencing……………………………………17 2.13 Amplicon-based next-generation sequencing………………………………18 2.14 Statistical analysis…………………………………………………………19 3. Results……………………………………………………………………………20 3.1 Design sgRNAs targeting Mlh1, Msh2 and their functional validation in vitro……………………………………………………………………20 3.2 Generate ICE simplex (multiplex) CRIM transgenic mouse………………22 3.3 Disrupt Mlh1, Msh2 in ICE CRIM mouse somatic cells and further characterize their molecular phenotypes……………………………………24 4. Discussion…………………………………………………………………………28 5. References…………………………………………………………………………32 | |
| dc.language.iso | en | |
| dc.title | 建立DNA錯配修復功能缺失的ICE CRIM小鼠模式 | zh_TW |
| dc.title | Generation of ICE CRIM mice deficient in DNA mismatch repair | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 潘思樺,游麗如,蘇怡璇 | |
| dc.subject.keyword | DNA 錯配修復,CRISPR/Cas9,組織特異性環化重組?,ICE CRIM,Mlh1,Msh2, | zh_TW |
| dc.subject.keyword | genome instability,DNA mismatch repair (MMR),Mlh1,Msh2,CRISPR/Cas9,Cre, | en |
| dc.relation.page | 105 | |
| dc.identifier.doi | 10.6342/NTU201702845 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-09 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 基因體暨蛋白體醫學研究所 | zh_TW |
| Appears in Collections: | 基因體暨蛋白體醫學研究所 | |
Files in This Item:
| File | Size | Format | |
|---|---|---|---|
| ntu-106-1.pdf Restricted Access | 6.65 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.