2020-05-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/714328摘要：本團隊在科技部計畫下所建立的”A4基因轉殖鼠核心設施”，運作已15年。本核心的競爭優勢與重要成果包括:做到”一站式客製化”服務，使用者只須提供基因名稱，本核心即完成並交付基因改造(Tg)鼠；具有提供兩種尖端技術”CRISPR/Cas9”及”胚胎幹細胞基因標的”的專業能力；本年度至Q3績效近1000萬元(43%,已逾35%部訂收支比標準, 較去年同期增長15%);本年新使用者佔56%，顯示需求度仍極高;國際基因轉殖技術學會會員，具專屬網站與國際接軌；持續獲得校配合款，並有AAALAC國際認證的本院動物中心全力配合。本核心設施期以台大的資源配合科技部服務全國，達到資源共享的理想。　　本核心本期(2/4; 四年期第二年)確切目標(權重)為提供服務(80%)、技術研發(10%)、合作研究(5%)及教育訓練/推廣(5%)。在提供服務方面，將產製各種品系(包括特殊品系如NSG)的基因改造鼠。在研發方面，主旨在優化CRISPR技術以提升服務效能及縮短製程(已完成的技術研發項目請見報告書及過去成果)，本期(2/4)將執行(i) 提升大片段DNA置入成功率與長度: 利用biotin-DNA模板及"Tild"法等；(ii)單/雙細胞期loxP遞送法；及(iii)利用最新發展的單細胞受精卵雙電擊法遞送上游及下游loxP，可提升loxP置入效率並縮短時程(新增); (iv)建立試管內Cre酵素剪除法鑑定cKO小鼠基因型(新增) (v)創造更精緻的動物模式，本核心改造NSG鼠作出擬人化小鼠及疾病工具鼠(共同發表Nat. Comm. 2020; 2020年洽談技轉中)可測試並開發PDX (patient derived xenograft)相關免疫藥物，下年度將產出可生產人類抗體的免疫缺陷鼠。在合作研究方面，本次提出2個合作研究，包括與呂理帆博士(UCSD, USA)及台大陳沛隆醫師合作(見四年計畫書)，將利用本核心開發的Tg鼠，研究調控型免疫T細胞在管控免疫耐受性的角色及開發出具有人類免疫基因的擬人化”主要組織相容性複合體”小鼠，以開創疾病動物模式的新紀元。在教育訓練/推廣方面已完成產業界及醫療院所說明會，成效亮眼(至Q3已達625人次)，未來將持續推動相關業務。　　本核心設施最終目標是成為我國醫藥生技的重要支援單位及提供基因轉殖鼠作為新藥研發的疾病模式的基石單位。<br> Abstract: The“A4 Transgenic Mouse Model Core” has been set up under the Ministry of Science and Technology's policy and operated with highest competitive technology for 15 years of prominent successes, including: Built as a "one-stop shop" facility for making knockout/Knock-in (Tg) mice; i.e., users simply provide the name of the gene, A4 core performs all the experiments and deliver the final Tg mice; One of a few that provides both sophisticated technologies, the “CRISPR/Cas9” and the “ES cell gene targeting” in one facility; Outstanding service income (till Q3) reaching NT$ 1000 millions (43%, higher than the “MOST” (Receipt/Expenditure) baseline 35%, 15% increase over last year); "new users" reaching 56% total users, indicating continuing high demands for A4 core’s expertise; ISTT (International Society of Transgenic Technology) members with core website available to global users; matching funds from NTU and Medical College and also the support of our AAALAC-accredited Animal Center. The A4 core facility is determined to implement these resources to serve our researchers, reaching the idea of resources sharing . We propose four aims of this fiscal year (2/4; the second year of four-year's grant) (workload proportions), including “Providing Tg services (80%); R & D (10%); Cooperative research (5%); and Education/ Dissemination (5%). For “Providing Tg services”, the A4 core will help generate all types of Tg mice in standard and special stains (e.g., NSG mice); for “R & D”, we aim at improving CRISPR technology to provide the best services and shorten the turn-around time for Tg production (see “Progress report” and “Previous achievement” for the established technologies). For this fiscal year (2/4), we will improve the CRISPR technology even further by executing (i) to use biotin-DNA template and "Tild method" developed in house for large DNA insertion; (ii) to set up strategy for One/Two cells loxP delivery for Tg mice production; (iii) to apply two consecutive electroporations on the same embryos to facilitate upstream and downstream CRISPR/loxP insertions (new proposal); (iv) In-vitro Cre excision of genomic DNA for cis/trans loxP determination (new proposal); (v) generating new NSG mouse models and disease models (Co-author, Nat. Comm. 2020; licensing under negotiation in 2020) for PDX (patient derived xenograft) and immune-check point inhibitor drug testing and drug delivery; For “Collaborative research”, we have proposed 2 collaborative projects, one with Dr. Lu (UCSD, USA) and the other, with Dr. Chen (NTU Hospital) (see our four year’s grant proposal, submitted in 2019), to use Tg mice lines generated by A4 core to dissect defined Treg cell subsets for maintaining immunological tolerance, and to generate revolutionizing humanized mice models for human disease study; and for “Education/Dissemination", the core has dissemination in biotech industry and hospitals with 625 participants (till Q3). We will continue to implement the relevant business. The ultimate goal of this core facility is to become an important supporting unit for medical biotechnologies in this national, and to strengthen the Tg mice being used as platforms for studying gene functions, and for new drug efficacy testing and validation.基因剔除/置入小鼠核心設施CRISPR/Cas9Transgenic/Knockout/knock-in micecore facilityCRISPR/Cas9