摘要:基因突變小鼠為研究人類基因功能、疾病機制及藥物研發的最重要的工具之一。本團隊的宗旨為建立任務導向的基因剔除或置入(knock-in)小鼠核心設施,以提供最尖端的技術協助研究者製造基因剔除/置入小鼠。本團隊自2005年建立(A4)基因轉殖鼠核心設施以來,已接受來自212位使用者提出的477個(500種)基因剔除/置入小鼠案件,至今(2015年11月)已產出並交付>356種可繼代遺傳的基因剔除/置入小鼠。近年最革命性的CRISPR/Cas9核酸酶法成為建構基因剔除小鼠最重要技術,本核心隨即發展該技術並能與國際團隊同步提供該服務,成為國內外可同時提供胚胎幹細胞法與CRISPR/Cas9核酸酶法的核心設施,本核心設施的特點是做到一站式服務,亦即使用者只須提供基因名稱及須求(剔除,置入、條件式剔除等),核心即負責所有技術至交付小鼠。本核心設施的目標是(1)以最前瞻的基因剔除/置入(KO/KI)技術,協助使用者研究基因功能、建立人類疾病動物模式、及完成藥物標的(druggable target)的確效驗證; (2)提供最具效率及最大效益的一站式基因轉殖鼠核心設施;及(3)強化學研界與生技產業的連結,增加國內學術及生技研發的競爭力。為達到上述目標,本核心設施未來一年的執行重點為1.繼續提供服務,佔80%,2.研發相關科技(佔5%),3. 合作研究計畫(佔5%),與4.教育訓練(5%)及推廣核心設施(5%)等。在重點1.繼續提供服務方面,本核心將同時提供胚胎幹細胞法與CRISPR/Cas9核酸酶法完成基因剔除/置入小鼠,包括條件式剔除/置入及特殊品系如免疫缺陷(如NSG, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ)小鼠的基因剔除/置入。本核心每年的委託案件約40-60個,自2015年提供CRISPR/Cas9服務後總案件增為104件(2015/1/1~2015/12/31),其中胚胎幹細胞法為37件,CRISPR/Cas9核酸酶法為67 件,顯示CRISPR/Cas9受到重視。此外,本核心的新使用者一直穩定成長,重復的使用者約佔25%,顯示本核心的需求度極高且持續增強。在重點2.研發相關科技(R&D)方面,未來一年將著重在提升CRISPR/Cas9技術的精準化及條件式基因剔除/置入的效率(詳見p.28)。有關R&D的進度,本核心已完成第一年(2015/5/1~2016/4/30)規劃的2項工作,包括以CRISPR/Cas9技術製作流程標準化(附件一)及開發擬人化小鼠疾病模式以提供個人化醫療更適切的評估工具,本核心已利用本校自Jackson Lab引進的NSG小鼠,並利用CRISPR剔除β2-microglobulin基因而產製出NSG-β2mnull小鼠,該小鼠因較不易產生移植對宿主排斥(GVHD)可大大提升人類細胞移植成功率,因該小鼠為本核心所開發,可分讓給國內使用者而無須受Jackson Lab的限制(目前已分讓予中研院學者);在3.合作研究方面,下年度將繼續第一年所規劃的與心臟科醫師合作,利用CRISPR進行多基因位點(3個基因)同時突變以建立國人常見的心因性猝死症候群小鼠模型以提供藥物研發,本核心目前已完成該基因突變小鼠,下年度將與使用者共同進行後續繁殖與分析。此外,本核心提出下年度與中研院生醫所陶秘華研究員合作進行”新型擬人化小鼠的開發”,將利用AAV攜帶人類細胞激素如Flt3l、GM-CSF及IL-4等基因,轉殖至NSG小鼠及NSG-β2mnull小鼠以測試其在重建人類免疫系統的成效,若成功,將來可提供國內用擬人化小鼠在感染症,癌症免疫療法等的相關研發。在4.教育訓練及推廣核心設施方面,本期(2015/5/1~今)共舉辦/參與13場說明會(包括外賓參訪),總教育及訓練人數達1217人次。此外,本核心與陳佑宗老師共同開發的螢光工具鼠本期分讓予2位國內學者(總分讓數達31個單位),且已有國際學者自Jackson Lab取得本小鼠(2014年分讓)後發表1篇論文(Cell Stem Cell, Dec., 2015 on line, IF=22.3,見內文);在國際使用者方面,本期運送4個品系(累計寄送>20個品系),且本團隊參與共同發表1篇高影響系數論文(Immunity, I.F.=21.56, July, 2015,見內文)。本核心的最終目標即是(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。
Abstract: Mouse has been one of the best tools for studying human gene function, disease mechanisms and drug discovery. The goal of this team is building up a project-based core facility to provide pioneer technologies to generate knockout/knock-in (KO/KI) mouse models for users. Since 2005 when the team started up the (A4) Transgenic Mouse Model Core facility team under governmental projects, it has received nearly 500 requests to KO/KI 477 genes from 212 users, and has successfully transferred > 356 germlined mouse lines to users. With the breakthrough of CRISPR/Cas9 technology in generating KO/KI mice, the team is able to cope with international core facilities and has provided the CRISPR/Cas9-based service to our users. This core has become a unique facility that can provide both ES cell-based and CRISPR/Cas9 nuclease-based gene KO/KI mice services. The powerfulness of the core is its “one-stop shop” service provider, i.e., from DNA to mouse, and users only need to give gene name and type of gene modifications (KO/KI/conditional KO), the core will make and deliver to users the ideal mice. The objectives of this core include (1) using pioneering KO/KI technologies to generate mice to help users in studying gene function, searching for druggable targets, and performing efficacy validation of candidate drugs; (2) to become a time-efficient and cost-effective one-stop shop core facility; and (3) to help strengthen the connection among academic, regulatory agency and biotech industry, and to promote our competitiveness and opportunities.To achieve above goals/objectives, this team proposes for the next fiscal year to achieve 4 aims: 1. continuing on providing services (80% of total effort), 2. performing Research and development (R&D) (5% effort) 3. Performing collaborative research (5% of effort), and 4. Executing education/disseminations (5%/5%). To aim 1. Continuing on providing services, we will provide both ES cell-based and CRISPR/Cas9 nuclease-based KO/KI mice services, including standard mouse strains and unique stains like immunodeficient (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, NSG) mice. We noticed that the average requested gene KO/KI cases is 40~60 per year. It has increased to 104 cases (20151/1~2015/11/30) since we provided CRISPR/Cas9 in 2015. Among them 37 were ES cell-based and 67 were CRISPR/Cas9-based KO/KI. Obviously, CRISPR has become an important technology. Besides, the number of new users continues to grow, indicating the popularity and importance of this core. To aim 2. performing Research and development (R&D), we will develop new (i.e. Cpf1) or modified Cas9 nuclease system to improve the accuracy and efficiency of nuclease-based gene KO/KI (see p.28 for detail). Our achievement for the first fiscal year (2015/5/1~2016/4/30) on the two proposed R&D subprojects has been successful. We have established standardization of operation (SOP) for CRISPR/Cas9 services (Appendix 1), and have used CRISPR/Cas9 to mutate the β2-microglobulin gene, in hope to reduce graft-vs-host-disease (GVHD), in NSG mice imported to our campus from the Jackson Lab, and generated the NSG-β2mnull mice which is more suitable for engrafting human cells. These mice can be shared more friendly to our users than the Jackson Lab (the mice have been transferred to Academia Sinica). To aim 3. Performing collaborative research, we will continue on what was proposed in the first fiscal year to collaborate with cardiology physicians to develop mouse models for sudden death syndrome using CRISPR/Cas9 to mutate 3 genes simultaneously. We have successfully made the mice with 3 mutations. In the next fiscal year we will help to breed the mice. In addition, we propose another collaborative research with Dr. MH Tao (IBMS, Acad. Sini.) to use AAV-Flt3, CM-CSF, IL-4 etc. (human cytokine/growth factor genes) to refine the NSG and NSG-β2mnull mice for better transplantation of human cells. This will allow us to provide better humanized mice for study of infection/host immunity, cancer relation immunotherapy etc. To aim 4. Executing education/ disseminations, we will continue on these activities. Our achievement on this category is very good. In the first fiscal year (2015/5/1-present), we have held and participated in 13 promotion activities including users/core communication/service dissemination activities and international user visit/promotion/seminars and total participants reached 1217. In collaboration with Dr. You-Tzung Chen, we have generated a double-florescent mouse line which has been shared with 31 Taiwan researchers. An international research team has obtained our mice from the Jackson Lab (donated in 2014) and published a high impact paper using the mice (Cell Stem Cell, 2015 Dec., on line, IF=22.3). Also, we have delivered 4 KO/KI mice (a total of > 20 strains) to international users and have resulted in a collaborative publication in “Immunity” (I.F.=21.56, 2015 July). The achievement of this core has been very successful. We hope to achieve our long term goals, i.e., to convince and facilitate our users to apply KO/KI mice as models for studying gene functions and disease mechanisms and for drug discovery to cure human diseases.