DNA-dependent protein kinase (DNA-PKcs)主要的功能在調控DNA雙股螺旋斷裂(double-strand break , DSB)的修復機制，當DNA雙股螺旋斷裂時會引起DNA-PKcs的自我磷酸化反應(autophosphorylation)來調控其本身活性。根據研究發現，當exon 58上的三個Thr2605、Thr2634及Thr2643磷酸位被alanine所取代，並且形成 DNA-PKcs3A/3A時，老鼠體內會有先天性骨髓(bone marrow)衰竭的現象產生。我的研究主要是育出帶有DNA-PKcs3A/3A BALB/c小鼠，期待能具備較高的擬人化(humanization)效力。在這個研究之前，我們嘗試通過直接注射Cas9、引導RNA (guide RNA)及模板DNA (donor DNA)至BALB/c小鼠受精卵中，但未能拿到我們所期望DNA-PKcs3A/3A BALB/c小鼠。因此，這次我們利用CRISPR/Cas9及G-418篩選之技術，在BALB/c胚幹細胞上進行基因編輯，主要是藉由標定於exon 58上之引導RNA誘導BALB/c胚幹細胞的DNA斷裂，並同時送入帶有DNA-PKcs3A、PGK:Neo及兩段同源序列的基因載體進行同源重組(homologous recombination)。接著，將帶有 DNA-PKcs3A之BALB/c胚幹細胞藉由囊胚注射至代理孕母小鼠中，期望孕育出帶有生殖系統傳遞能力(germline transmission)的嵌合體(chimeric)小鼠，最後，藉由配種孕育出帶有DNA-PKcs3A/3A的子代。人類CD34+細胞是一種造血幹細胞，具有在免疫缺陷小鼠身上建立人類免疫系統之能力，我們期望具有先天性骨髓衰竭及免疫缺陷特性之DNA-PKcs3A/3A BALB/c小鼠，可以提升人類CD34+細胞建立擬人化老鼠(humanized mice)的能力並且作為人類免疫系統研究之平台。一旦成功，我的研究將對藥物測試和人類疾病這些研究領域產生極大的貢獻。

DNA-dependent protein kinase (DNA-PKcs) is essential for DNA repair pathways. The activity of DNA-PKcs is regulated by different phosphorylation sites in response to DNA double strand breaks (DSBs). It was reported that three alanine substitutions at the Thr2605 phosphorylation cluster, including Thr2605、Thr2634 and Thr2643, on exon 58 (DNA-PKcs3A/3A) would cause early bone marrow failure in homozygous mice. This study is aimed to generate DNA-PKcs3A/3A mutant mice in the BALB/c background and to evaluate its potential for humanization. A previous attempt by direct injection of Cas9 plasmid, guide RNA, and donor DNA into BLAB/c fertilised eggs failed to generate the desired mutant. Thus an alternative approach by combining CRISPR/Cas9 and neo-based screening on BALB/c embryonic stem cells is taken. Specifically, we use Cas9 and exon 58-targeting guide RNAs to induce DNA breaks in BALB/c embryonic stem cells. At the same time we supply a mutant template of DNA-PKcs3A within a cassette that has the PGK:Neo selectable marker for positive selection and two flanking arms for homologous recombination. In the future, the selected DNA-PKcs3A mutant knock-in ES cells will be injected into blastocysts to produce chimeric mice by foster mothers. Hopefully these mutant mice will have the ability of germline transmission to generate a line of homozygous DNA-PKcs3A/3A offsprings after further breeding. These mice will be evaluated whether they can serve as a better platform for humanization by human CD34+ cells. Human CD34+ cells are hematopoietic stem cells that have been known to be able to establish the human immune system in immune compromised mice. With expected early bone marrow failure and immune deficiency in homozygous DNA-PKcs3A/3A mice, we hope these properties could augment the performance of CD34+ cell-based humanization and serve the need of an excellent platform for establishing the human immune system. Once succeeded, my work will have great contributions to drug testing and disease research that require faithfulness to bona fide human immunity.

中文摘要………………………………………………………………………………………i
英文摘要……………………………………………………………………………………ii
目錄……………………………………………………………………………………………iv
圖目錄………………………………………………………………………………………viii
第一章、緒論…………………………………………………………………………………1
1.1、前言…………………………………………………………………………………1
1.2、擬人化老鼠(humanized mice)………………………………………………………2
1.3、免疫缺陷鼠(immunodeficient mice)………………………………………………3
1.3.1、SCID mice………………………………………………………………………3
1.3.2、NOD mice………………………………………………………………………4
1.3.3、NSG (NOD/SCID/IL2Rγnull) mice………………………………………………5
1.4、interleukin-2受體(IL-2R ) …………………………………………………………5
1.5、DNA-dependent protein kinase catalytic subunit (DNA–PKcs) ……………………6
1.6、胚幹細胞（embryonic stem cells）…………………………………………………7
1.7、小鼠胚幹細胞之體外建立…………………………………………………………7
1.8、基因组编輯(genome editing) ………………………………………………………8
1.9、CRISPR-Cas…………………………………………………………………………9
1.9.1、CRISPR-Cas9之起源…………………………………………………………9
1.9.2、CRISPR- Cas9之防禦機制……………………………………………………10
1.9.3、第二型CRISPR/Cas -CRISPR-Cas9…………………………………………10
1.9.4、CRISPR-Cas9基因工程應用…………………………………………………11
1.10、轉染(Transfection) ………………………………………………………………12
1.10.1、脂質體介導(lipofection) ……………………………………………………12
1.10.2、Fugene………………………………………………………………………13
1.10.3、電穿孔法(electroporation) …………………………………………………13
1.11、G-418篩選(G-418 selection)……………………………………………………13
1.12、Cre/loxP重組(Cre-LoxP recombination) ………………………………………14
1.13、囊胚注射(blastocyst injection) …………………………………………………14
1.14、研究方向…………………………………………………………………………16
第二章、材料與方法…………………………………………………………………………17
2.1、BALB/cJ line I胚幹細胞之建立…………………………………………………17
2.1.1、纖維母細胞培養液之置備……………………………………………………17
2.1.2、Primary MEFs之製備…………………………………………………………17
2.1.3、MEFs飼養層細胞(feeder layer)之製備………………………………………18
2.1.4、細胞株來源……………………………………………………………………18
2.1.5、幹細胞培養液之製備…………………………………………………………18
2.1.6、細胞繼代之步驟………………………………………………………………19
2.1.7、細胞冷凍之步驟………………………………………………………………19
2.1.8、細胞解凍之步驟………………………………………………………………19
2.1.9、細胞冷凍液之製備……………………………………………………………20
2.2、細胞計數之步驟……………………………………………………………………20
2.3、源基因轉染之步驟…………………………………………………………………20
2.3.1、Lipofectoamine® 3000 Transfection Reagent…………………………………20
2.3.2、FuGENE® HD Transfection Reagent…………………………………………21
2.3.3、Neon® Transfection system……………………………………………………21
2.4、G-418篩選之步驟…………………………………………………………………22
2.5、細胞stable colones挑選之步驟…………………………………………………22
2.6、細胞DNA之萃取之步驟…………………………………………………………23
2.7、洋菜凝膠電泳(agarose gel electrophoresis) 之步驟………………………………23
2.8、聚合酶連鎖反應(Polymerase chain reaction,PCR)之步驟………………………24
2.9、Prkdc-loxPneo BALB/c mESCs基因之PCR分析設計…………………………24
2.10、TA cloning之步驟………………………………………………………………25
2.11、質體DNA萃取之步驟…………………………………………………………27
2.12、Probe標定DIG-dUTP之步驟…………………………………………………27
2.13、南方墨點法(Southern blot)之步驟………………………………………………28
2.14、DNA定序(DNA sequencing)之步驟……………………………………………31
2.15、黴漿菌檢測(mycoplasma detection)之步驟……………………………………31
第三章、實驗結果……………………………………………………………………………33
3.1、飼養層細胞(feeder layer)之製備…………………………………………………33
3.2、BALB/cJ line I胚幹細胞建立……………………………………………………33
3.3、比較外源基因轉染技術之轉染效率………………………………………………34
3.4、pPrkdc-loxPneo載體之設計………………………………………………………34
3.5、轉染pPrkdc-loxPneo載體至BALB/c mESCs……………………………………35
3.6、Prkdc-loxPneo BALB/c mESCs之基因型分析genotyping………………………35
3.7、Prkdc-loxPneo BALB/c mESCs之南方墨點法(Southern blot) …………………36
3.8、DNA定序(DNA sequencing) ……………………………………………………36
3.9、黴漿菌檢測(mycoplasma detection) ……………………………………………37
第四章、討論…………………………………………………………………………………38
第五章、參考文獻………………………………………………………………………41

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