2018-05-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/695994摘要：本核心運作邁入第13年，為“一站式客製化”且同時提供CRISPR/Cas9核酸酶法及胚胎幹細胞基因標的技術建構基因轉殖小鼠的設施。使用者只須提供基因名稱及須求，本核心即完成所託交付基因剔除/置入小鼠。統計2015.5-2017.12為211件，包括124件核酸酶法及87件胚胎幹細胞法。核心每年委託案件40~60件且逐漸成長，本年度Q3季已達67件，服務收入預估可達1千萬。國際使用者約佔5%，而“新使用者”穩定成長約佔年總數50%，顯示設施需求度極高。本核心於使用者委員會的督導下，秉持“first-come-first-serve”的原則完成服務，同時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/)，與國際接軌。 未來計畫確切目標為提供服務(80%)、技術研發(10%)、合作研究(5%)及教育訓練及推廣(5%)。執行重點在優化核酸酶法服務內容。包括(i)以體外受精(IVF)配合冷凍胚技術供應所需的受精卵，達到充足卵量及擴大實驗自由度；(ii)以受精卵測試並選擇效率最佳的sgRNA以提高成功率，預計Q1季提供服務；(iii)以電擊穿孔法於IVF受精卵進行CRISPR，成功後將增列為服務項目。在1.提供服務，將產製包括條件式、多基因剔除和特殊品系如免疫缺陷小鼠(NSG mice)的基因剔除/置入小鼠等。在2.技術研發，將包括提升核酸酶法作用效率與降低插入/缺失突變機率;新型單股DNA模版提升大片段基因置入的成功率;電擊穿孔法提升loxP置入效率及開發新型擬人化小鼠以優化服務。在3.合作研究，將與國際免疫學專家呂理帆博士(UCSD,USA)合作，以特異的工具鼠條件式剔除三種不同次族群的調控T細胞，解析其免疫調節功能，該工具鼠亦將分讓國內使用。雙方合作多年且共同發表亮點論文(Immunity 2015, IF>20)，另有論文於Nature系列雜誌審稿中。另將與中研院陶秘華研究員合作測試以AAV載體表達人類細胞激素達到優化擬人化小鼠，提供國內機構進行PDX篩選抗癌藥物的平台。在4.推廣與教育訓練，去年總人數達2262人次，且已分讓小鼠予43個研究者及Jackson Lab，成效亮眼，未來將繼續執行。 本核心終極目標是成為醫療生技的重要支援單位及強化以基因轉殖鼠作為新藥研發的疾病模型。<br> Abstract: The A4 Transgenic Mouse Model Core (TMMC) facility has now been operating for 13 years and becomes an "one-stop shop" service for making KO/KI mice using both the CRISPR/Cas9-based and the ES cell-based technology; i.e., users simply provide the name and requirements of the gene, TMMC will perform all the experiments and deliver the final KO/KI mice to users. The total number of cases from 2015/5~2017/12 is 211, including 124 CRISPR and 87 ES cell cases. TMMC receive about 40~60 requests per year and the demand grows each year. We have received 67 cases by the Q3 of year 2017, estimated service revenue will reach NT$10 million. TMMC have around 5% international users. The annual "new users" of TMMC has been steadily growing by about 50% totals, indicating that this core’s demand is extremely high. Under the guidance and assistance of the user committee, TMMC has always been the first-come-first-serve principle to complete the service cases. TMMC has its own website (http://140.112.133.74/) which gives the ability to be international. This grant proposal has the following objectives: Providing services (80%); R&D (10%); Cooperative research and Education/disseminations (5%). Include (i) using IVF coupled with embryo freezing/ thawing technologies to supply substantial amount of fertilized eggs for CRISPR. (ii) Applying in-vivo testing of the efficiency and select the most efficient sgRNAs for CRISPR; (iii) using electroporation of embryos for CRISPR. We expect to open the service by the Q3 season of the next fiscal year, if funded. In 1., Providing service, TMMC will provide the classical and conditional KO/KI in standard and special stains of mice (e.g., immunodeficient mice) and for simultaneous multiple gene KO/KI services; 2., R&D: we will optimize service technologies; including develop technology to facilitating CRISPR success rate; improving the inserting efficiency of large DNA into genome by using a single-strand DNA template manufacturing method; using electroporation method to target the loxP sites for cKO mice; and making novel humanized NSG mice; 3. Cooperative research, we will collaborate with Dr. Lu and focus on utilizing tool mice that provide conditional deletion of 3 specific Treg cell subsets for dissecting the role of a defined Treg cell subset. We and Dr. Lu have produced fruitful outcomes (Immunity, 2015, IF>20; one more under review at Nature series). The other is collaborating with Dr. Tao to improve humanized mouse models. We propose to KO murine MHC class I/II on our NSG-b2m KO mice to generate dKO mice for transplantation of human immune cells. To establish the humanized PDX more delicate, Dr. Tao will use AAV vector to deliver human cytokines genes; and in 4., Our achievements have been remarkable (the total no. of business promotion activities/education/training has reached 2,262 persons, sharing mice to 43 domestic and international researchers and to Jackson Lab). We will continue to implement the relevant business. The ultimate goal of this core is to become an important supporting unit for medical biotechnologies, and to strengthen domestic use of KO/KI mice for the development of new drugs.基因剔除/置入小鼠核心設施CRISPR/Cas9Transgenic/Knockout/knock-in micecore facilityCRISPR/Cas9