2017-05-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/701711摘要：基因轉殖/剔除小鼠為研究人類基因功能、疾病機制及藥物研發的最重要工具之一。有鑑於此，在科技部與本校的計畫補助下，本團隊成立了服務全國的基因轉殖鼠核心設施 (TMMC)，本研究計畫的目的是在爭取本團隊建立的基因轉殖鼠核心設施的持續維運。本核心設施的宗旨為建立”一站式客製化”的基因剔除/置入(knock-in)小鼠核心設施，提供最尖端的技術協助研究者製造基因剔除/置入小鼠。本團隊建立的核心設施自成立迄今11年(2005年5月~2017年2月)，每年的委託案件約40~60件，目前完成並交付405種成功繼代遺傳的基因剔除/置入小鼠。本核心設施已成為國內少數可同時提供CRISPR/Cas9核酸酶法及胚胎幹細胞基因標的技術建構基因改造小鼠的核心設施，一站式服務是本核心設施的最大特點及優勢，使用者只須提供基因名稱及須求(剔除，置入、條件式剔除等)，本核心設施即可完成所託而交付基因剔除/置入小鼠。自2014年提供CRISPR/Cas9技術後案件數逐漸增加，統計2015/5/1~2017/1/31的總服務案件數為131件，其中CRISPR/Cas9核酸酶法為81件，胚胎幹細胞法為50件，顯示CRISPR/Cas9受到重視。本核心在使用者委員會的協助督導下(歷次會議記錄見附件)，一直秉持”first-come-first-serve”的原則完成服務案件，同時，也在本校基因體中心及醫學院提供的配合款資源下，建置完善的軟硬體設備與專屬網站(http://140.112.133.74/)，提供國內外使用者諮詢。在國際使用者方面，本期已運送13個品系，累計國際使用者達4%，地點擴及歐、亞洲及美國。本核心每年的”新使用”者一直穩定成長約佔55%，顯示需求度極高且持續增強。本計畫書提出本核心設施下期計畫的確切目標包括1.提供基因剔除/置入服務(80%)，2.技術研發(R&D) (10%)，3. 合作研究(5%)及4.教育訓練及核心設施推廣(5%)。在1.提供服務方面，本核心設施同時提供CRISPR/Cas9核酸酶法與胚胎幹細胞法技術產出基因剔除/置入小鼠，包括條件式剔除/置入，多基因同時剔除和特殊品系如免疫缺陷(如NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, NSG)小鼠的基因剔除/置入及大片段基因置入服務等; 在2.技術研發方面，將著重在提升CRISPR/Cas9剔除/置入的效率，尤其是大片段基因置入的效率; 在3.合作研究方面，本核心設施提出與國際學者呂理帆博士(UCSD, USA)及本院心臟科醫師合作，呂博士為mirRNA在免疫學角色的專家，雙方合作多年且共同發表亮點論文(Immunity 2015, IF=21.6)，有助提升本核心設施的國際知名度。未來一期合作內容是完成特異的工具鼠，可精準且條件式剔除(conditional KO)三種不同次族群(cell subset)的調控T細胞(Treg)，提供解析特定次族群的調控T細胞在維持免疫耐受性扮演的角色，完成的工具鼠亦將分讓國內使用，有助連結國內與國際研究接軌。另一合作研究為建立國人常見的心因性猝死症小鼠模型以提供藥物研發，本核心已利用CRISPR/Cas9完成此疾病模型，未來一期的合作內容是協助使用者(本院心臟科醫師)進行後續繁殖、基因型分析及表徵探討。在4.教育訓練及核心設施推廣方面，本核心過去成效亮眼(譬如核心業務推廣活動與教育訓練總人數達1694人次，分讓基因轉殖小鼠予國內外43個研究者及國際知名動物設施Jackson Lab，見計畫書內文)，下期亦將繼續執行相關業務。本核心設施的最終目標為(1)成為國內醫療生技的重要支援單位，(2)強化國內利用基因剔除/置入小鼠作為研究人類基因功能與疾病的平台及作為新藥研發的疾病動物模式。<br> Abstract: Transgenic/knockout mice have been one of the best tools for studying human gene function and diseasemechanisms, and for drug discovery. Because of this, we have founded a transgenic mouse model core(TMMC) facility available for national users, under grants from the Ministry of Science and Technology(MOST), and National Taiwan University (NTU). This proposal is to request continuing and sustainedfunding to the TMMC facility. The goal of TMMC is to build up a “one-stop shopping” service corefacility; users only need to inform the core of the gene and type of modification (KO/KI/conditional KO)they require, they will receive the ideal mice from TMMC without extra effort of their own. Oversight byMOST and NTU, TMMC has grown to be a unique facility that can provide both CRISPR/Cas9nuclease-based and ES cell-based gene KO/KI mouse services. Below is the summary of the achievementof the TMMC facility. Over the past 11 years (2005/5-2017/2) the TMMC has received 40~60 annual KO/KIrequests and has transferred 405 KO/KI germlined mice to users. During 2015/5-2017/2, TMMC has 131requests with 81 CRISPR-based and 50 ES cell-based KO/KI. Obviously, CRISPR has dominated recently.Under the supervision of the users’ committee (see appendix for meeting minutes), TMMC has followed theservice guideline of “first-come-first-serve”. Besides, with the resources supplied by NTU College ofMedicine and NTU Center for Genomic Medicine, TMMC has comprehensive hardware and software, andruns independent website http://140.112.133.74/ for national and international users. Our international users,representing ~4% of users, encompass Europe, USA and Asia. The achievement of TMMC has been veryvisible. More importantly, the number of first-time users continues to grow ~55% each year, indicating thepopularity and the need of TMMC.This competing grant application proposes to execute the following specific aims: (1) Providing KO/KItechnical services (80% effort); (2) Performing research and development (R&D) (10%); (3)Performing collaborative research (5%); and (4) Executing/disseminating education (5%). In (1)Providing KO/KI technical services, TMMC will provide both CRISPR-based and ES cell-based KO/KImouse services for generating classical and conditional KO/KI, single and multiple point mutations onroutine (e.g. C57BL/6) and unique mouse strains like NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/ SzJ) mice; in (2)Performing R&D, we will modify the CRISPR system to improve the accuracy and efficiency of KO/KI oflarge fragments; in (3) Performing collaborative research, we propose to continue our collaborations with Dr.Li-Fan Lu (UCSD, USA) and our institutional cardiologist team. We and Dr. Lu, a miRNA in immunologyexpert, have produced a fruitful outcome (Immunity, 2015, IF=21.6). Our future collaboration will focus ongenerating tool mice that can provide temporally and respectively deletion of 3 specific Treg cell subsets fordissecting the role of a defined Treg cell subset for maintaining immunological tolerance. We hope thiscollaboration will earn the TMMC its international publicity. For the cardiology collaboration, we have usedCRISPR technology to generate a mouse model of human Brugada’s sudden death syndrome with 3mutations prevalently found in Taiwanese patients. We propose to continue our collaboration by providingbreeding and genotyping expertise for this complex disease model; in (4) Executing disseminating education,we will continue performing these activities/services. Our achievement for the past fiscal year has beensuccessful (we have held many promotion activities with total participants reaching 1694, and we havedistributed many Tg/KO lines to 43 investigators including one to the Jackson Lab. (See appendix for detail).We hope to be able to continue to achieve our long term goals, i.e., (1) to become a strong support unitfor the biomedical industry in this nation; and (2) to convince and facilitate our users to apply KO/KI mice asmodels for studying gene functions and disease mechanisms and for drug discovery to cure human diseases.基因剔除/置入小鼠核心設施CRISPR/Cas9Transgenic/Knockout/knock-in micecore facilityCRISPR/Cas9