核心课程遗传工程与悉生动物模型

1、THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES遗传工程与悉生动物模型遗传工程与悉生动物模型Genetic Manipulated and Human flora-associated Genetic Manipulated and Human flora-associated (HFA) Animal Models(HFA) Animal Models 生命科学核心课程王 勇第三军医大学基础部实验动物学教研室E-mail:Tel:753882THIRD MILITARY MEDICAL UNIVERSITYC

2、OLLEGE OF BASIC MEDICAL SCIENCESContents pBackgroundpGentically Manipulated AnimalspHuman Flora-Associated(HFA) AnimalsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESintroductionTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES THIRD MILITARY MEDICAL UNIVERSITYCOL

3、LEGE OF BASIC MEDICAL SCIENCESHow to understand this superorganism?疾病动物模型研究需求针对生命科学关键问题疾病动物模型无菌技术遗传工程技术解析机体自身(宿主)基因组功能解析机体寄生菌群(宏)基因组能生长发育健康疾病与菌群关系密切Science.2012;336THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES第一章第一章 遗传工程动物遗传工程动物Genetically manipulated animalsTHIRD MILITARY MEDIC

4、AL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES利用模式动物研究基因与疾病关系的两利用模式动物研究基因与疾病关系的两种模式种模式THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp反向遗传学反向遗传学（reverse genetics）：从基因到表型：从从特定基因的改造特定基因的改造到到整体动物表型整体动物表型分析：分析：通过向动物基因组增加一个基因（转基因）或通过向动物基因组增加一个基因（转基因）或删除一个基因（基因敲出），观察动物表型变化删除一个基因（基因敲出），观

5、察动物表型变化，进而分析基因与表型的关系，研究基因功能，进而分析基因与表型的关系，研究基因功能THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp优点：能深入、系统地研究特定基因功能，能在较短能深入、系统地研究特定基因功能，能在较短时间获得详实的研究资料时间获得详实的研究资料p缺点：动物表型往往受多基因调控，一个基因改变不动物表型往往受多基因调控，一个基因改变不能反应生物学机制的全貌能反应生物学机制的全貌单纯的表型分析往往会单纯的表型分析往往会“漏掉漏掉”一些主要信息一些主要信息THIRD MILITARY ME

6、DICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp正向遗传学(Forward Genetics)：从表型到基因 从特定表型改变到基因分析：通过高效率的、高通量的通过高效率的、高通量的随机诱变技术随机诱变技术大规模大规模制备突变突变动物，然后从大量的基因突变动制备突变突变动物，然后从大量的基因突变动物中筛选物中筛选表型变异个体表型变异个体，最后通过遗传学分析，最后通过遗传学分析、染色体定位、分子克隆等手段，最终克隆、染色体定位、分子克隆等手段，最终克隆导导致表型变异的突变基因致表型变异的突变基因THIRD MILITARY MEDICAL UNI

7、VERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp优点：优点：可获得较完整的遗传信息可获得较完整的遗传信息p缺点：缺点：遗传分析与相关基因克隆是一繁琐过程遗传分析与相关基因克隆是一繁琐过程需要大规模饲养动物，成本高需要大规模饲养动物，成本高THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES第一节第一节 利用反向遗传学模式研究利用反向遗传学模式研究基因功能基因功能THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCI

8、ENCES利用正向遗传学研究基因功能的基本思路利用正向遗传学研究基因功能的基本思路文献查阅、课题设计确定候选基因RNA导向的核酶技术(CRISPR/Cas9)基于DNA同源重组的基因敲出基于位点特异性核酶(ZFN,TALEN)的基因敲出基因敲出（knock-out)动物模型系统、综合的表型分析正常基因转入基因回复突变动物模型确定基因功能THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES一、基因敲出动物一、基因敲出动物(Gene knock-out animal)THIRD MILITARY MEDICAL UNI

9、VERSITYCOLLEGE OF BASIC MEDICAL SCIENCES候选基因确定候选基因确定p依据课题目标，解析课题目标所涉及的生物学过程p梳理生物学过程中所涉及的可能信号通路（网络）或分子机制p选择关键节点中的关键基因THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES全球性的规模化基因敲出计划全球性的规模化基因敲出计划-initiative to knockout every geneTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL

10、SCIENCESHow to knock out a gene？在Evan等教授建立胚胎干细胞的基础上，上世纪80年代由Capecchi, Smithies 等建立了基于胚胎干细胞(ES细胞)DNA同源重组的基因敲除技术 Smithies Capecchi EvanShared 2007 Nobel prize in Medicine and PhysiologyTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES原原 理理pStep1在在ES细胞中通过细胞中通过DNA同源重组同源重组，将已发，将已发生失活突变的基

11、因打靶载体置换基因组生失活突变的基因打靶载体置换基因组内野生型的目标基因位点，使目标基因内野生型的目标基因位点，使目标基因活性消失活性消失 Oliver Smithies and Mario R. CapecchiTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES外显子外显子785687Neo5TKNeo敲除载体野生拷贝基因敲除拷贝：基因敲除拷贝：失去正常结构或功能失去正常结构或功能同源重组基因敲除的原理图基因敲除的原理图578THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF

12、 BASIC MEDICAL SCIENCES双向筛选获得基因敲出的双向筛选获得基因敲出的ES细胞细胞1234TK2NeoNeoG418 抗性GANC抗性1.1.同源重组同源重组2TKNeo1234NeoTKNeo2214G418 抗性GANC敏感2.随机整合3.没整合G418 敏感GANC抗性THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpStep2将发生将发生同源重组的同源重组的ES细胞输入囊胚，建立细胞输入囊胚，建立堪合体胚胎，进而获得堪合体动物。堪合体胚胎，进而获得堪合体动物。 Martin J. E

13、vans THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpStep3让堪合体动物与野生型动物的交配，通让堪合体动物与野生型动物的交配，通过其过其源自外源源自外源ES细胞的配子细胞的配子获得基因敲获得基因敲出的杂合体动物，进而获得纯合体动物出的杂合体动物，进而获得纯合体动物THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESStep1Step2Step3Summary of this technologyTHIRD MILITARY

14、 MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESCharacteristics of knockout(KO) animalsp KO小鼠的所有细胞，包括生殖细胞，都缺乏同一特异基因表达p KO动物可同时从DNA、RNA、蛋白质、细胞、组织、器官、整体动物等多种水平直接，动态地观察基因在整体动物内的活动情况以及改变该基因后引起的表型变化.p 若靶基因为发育或生长的必须基因，往往会导致动物发育异常或过早死亡THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESCo

15、nditional knock-outp在特定的细胞、组织或器官中特异性敲出靶基因，而在其他细胞、组织或器官中靶基因未发生功能缺失p是目前克服常规基因敲出技术缺陷的主要手段THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp位点特异性重组酶-条件性基因敲出的必备工具Cre: Create recombination enzyme在噬菌体中发现，可引起长36 bp的特殊顺序（loxp， location of cross) 间的重组，从而敲除 loxP 间的DNA。 Gu Hua于1995 年首先将其应用在小鼠基因

16、敲除中。THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESloxp sites in the same orientationloxp sites in the same orientationloxp sites in opposite orientationloxp sites in opposite orientationCreCre酶的作用模式酶的作用模式THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESThe princi

17、ples of conditional gene knock-outloxp AlleleTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESLoxp-Allele miceTissue-specific Cre transgenic miceTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESEngineered Nuclease technology-适用于不同物种的基因敲出技术适用于不同物种的基因敲出技术pClassic gene

18、 knock-out technology:高度依赖于永生化、全能性的胚胎干细胞高度依赖于永生化、全能性的胚胎干细胞流程繁杂、周期长，不适用于时代间隔长流程繁杂、周期长，不适用于时代间隔长的大动物的大动物THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpCurrently available engineered nuclease technologies:ZFN(Zinc Finger Nuclease)TALEN(TALE-like effector Nuclease)CRISPR/Cas9(Cluster

19、ed Regularly Interspaced Short Palindromic Repeat/ CRISPR-associated System)THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESZFN-mediated genomic editionpZFN: zinc-finger nuclease,锌指核酶是人工构建的序列特异性核酸内切酶，由两部分组是人工构建的序列特异性核酸内切酶，由两部分组成：具备高度序列特异性的成：具备高度序列特异性的DNA结合功能域结合功能域和不具和不具备序列特异性的备序列特异

20、性的DNA内切酶功能域内切酶功能域其中，其中，DNA结合功能域由结合功能域由3-4个锌指结构域个锌指结构域组成，每组成，每个锌指结果识别个锌指结果识别3bp，则一个锌指核酶识别，则一个锌指核酶识别9-12bp一个一个ZFN分子指切断一条分子指切断一条DNA链链THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESZFN介导介导gene knock-out的原理的原理p 由两个互补的ZFN分子（左向ZFN分子ZFN-L、右向ZFN分子ZFN-R）同时特异性地与靶位点结合，在靶位点导入双链断裂切口（Double stra

21、nd break，DSB）p 由于DSB的存在启动了细胞基因组修复机制，由此可实现多种位点特异性遗传修饰： 细胞通过错配率很高的（细胞通过错配率很高的（error-prone）“非同源性末非同源性末端连接端连接”（Non-homologous End Joining,NHEJ）机）机制修复制修复DSB，在，在ZFN靶位点造成靶位点造成碱基插入或缺失碱基插入或缺失，形成，形成位点特异性突变，导致靶基因功能缺失位点特异性突变，导致靶基因功能缺失 若在修复若在修复DSB时胞内存在与靶位点同源的时胞内存在与靶位点同源的DNA片段，则片段，则细胞主要通过细胞主要通过DNA同源重组（同源重组（Homolo

22、gous Recombination, HR）的机制修复的机制修复DSB，由此可实现靶，由此可实现靶基因置换或校正（基因置换或校正（gene replacement or correction）以及外源以及外源DNA片段定点敲入（片段定点敲入（gene knock-in or addition）THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESZFN-mediated gene knock-out and knock-inTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BA

23、SIC MEDICAL SCIENCESZFN-mediated gene knock-out in animals-Science, 2009, 325:433The first gene knock-out rats generated by ZFNTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESKnock-out pig generated by ZFNTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESZFN technol

24、ogy: primarily designed for human gene therapyTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESCCR5 mutagenesis and disablement in T cells ex vivo by ZFN for HIV patients-A phase I clinic trial has been finishedTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES-Natur

25、e, 2011In Vivo targeted gene insertion using ZFNTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESZFN技术的关键技术的关键p筛选最佳的锌指结构域组合（Zinc Finger Arrays）是形成特异性高、亲和力强的DNA结合功能域的关键p不是每一个依照靶序列组合的Zinc Finger Array都有活性，由此限制了ZFN技术适用性THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCEST

26、ALEN technologypA more powerful genomic editing tool, which is moving over ZFN理论上，针对任何基因的任何序列均可构建有理论上，针对任何基因的任何序列均可构建有活性的活性的TALENTALEN分子分子脱靶效应低于脱靶效应低于ZFNZFN，细胞毒性较弱，细胞毒性较弱目前正作为一种常规的基因修饰技术在迅速推目前正作为一种常规的基因修饰技术在迅速推广和应用，大有替代广和应用，大有替代ZFNZFN和和RNAiRNAi技术之势技术之势THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASI

27、C MEDICAL SCIENCESThe TALEN working mechanismp TALE（Transcription activator -like effectors）是一种源于植物致病菌Xanthomonas的转录激活因子，用于在细菌感染宿主细胞时启动宿主基因表达、以促进细菌感染p 研究发现，TALE因子与宿主基因靶位点的结合是通过一段模块重复序列（Modular repeats array)实现的，一个模块识别一个碱基，模块与碱基之间呈一一对应的关系p 每个模块重复序列约34个氨基酸，模块之间除了第12和13位氨基酸不一样以外，其余氨基酸序列几乎一致；而这两个不一致的两个氨

28、基酸决定了模块识别碱基的特异性，被称为RVD（repeat variable di-residues)THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTALETALE特异性识别碱基特异性识别碱基的原理：的原理：TALEs由数10-30个特异性识别DNA碱基的串联“重复模块”和两侧的N-末端及C-末端序列组成（如上图a）。每个“重复模块”包含34个氨基酸，第12和13位残基是靶向识别的关键位点，被称作重复可变的di-residues（RVDs）位点。TALEs上的四种RVDs（如图b）分别识别四种碱基。THIRD

29、 MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESRe-assemble of TAL repeat unitsRe-assemble of TAL repeat unitsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTALENTALEN的特异性打靶的原理（同的特异性打靶的原理（同ZFNZFN）：）：将特异性识别靶基因的TALE蛋白与ForKI内切酶的核酶功能域相融合，构建成融合分子TALEN(TALE-nuclease，如上图); 将

30、2个可与靶位点上下游互补链特异性结合的TALEN分子同时结合在靶基因位点，每个TALEN分子切断一条DNA链，两个TALEN则可在靶基因位点形成DNA双链断裂（double-strand break，DSB），引入的DSB可激活DNA自我修复，从而引起基因的突变和促进靶位点DNA同源重组。THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTALEN基因敲出原理THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTALEN TALEN

31、技术的发展历程技术的发展历程THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES利用利用TALEN技术研制的基因敲出动物技术研制的基因敲出动物Science, 2011,29:695THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESNature Biotechnology, 2013,31:23THIRD

32、 MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES首次报道了利用TALEN技术创制了基因敲出大动物（猪、牛）-PNAS,2012,109:17382THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESThe list of TALEN knock-out animals is rapidly growing.pRabbitpSheeppGoatpMonkey.TALEN is a powerful tool to knockout genes

33、including but not limited to mice! Just work with it!THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESCRISPR/Cas9 system-currently, the most recently genomic editing toolpCRISPR/Cas9(clustered regularly interspaced short parlindromic repeats/CRISPR-associated system9)a RNA-guided DN

34、A degradation system, originally the bacterial defense system against invading foreign nucleic acids THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESBacterialEukaryotic organismThe bacterial CRISPR/Cas9 system is somewhat like the eukaryotic RNAi system. The main difference is: CRI

35、SPR/Cas9 targets DNA, and RNAi targets RNA. This is what matters! THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpThe actual CRISPR/Cas9 system working in mammalian cells is rather simple:Human codon-optimized Cas9-encoding geneA small guiding RNA(sgRNA)gRNA and Cas9 protein assem

36、bled to be functional RNA-guided nuclease in cellsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESThe working CRISPR/Cas9 system in mammalian cells-Science,2013,339: 821THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESStructure of Cas9 and sgRNATHIRD MILITARY MEDIC

37、AL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESThe binding of Cas9-sgRNA complex to target site is PAM-dependentpPAM is the tri-nucleotides “NGG” adjacent to the sgRNA-guiding sequence on target siteTHIRD MILITARY MEDICAL UNIVERSITYCOL

38、LEGE OF BASIC MEDICAL SCIENCESIncreased genomic editing specificity using Cas9 nickasepBy inactivating one DNA cleavage domain of Cas9 protein, Cas9 protein can be mutated to be nickase capable of cutting one DNA strand.pBy using two sgRNAs binding to adjacent site located on opposite strands, Cas9/

39、sgRNA-mediated gene editing can be largely more specific. THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESCRISPR/Cas9-mediated genomic editing in aniamlspThe CRISPR/Cas9 system has been used in many species:Mice Pigs

40、MonkeysRats THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpGene-modified monkeys via CRISPR/Cas9 systemTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESGenomic-editing test in Monkey cellsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESOne-step

41、generation of double gene-KO monkeysTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpGene-modified pigs via CRISPR/Cas9Using a sgRNA targeting the exon2 of pig NPC1L1, we achieved genomic editing in pigs at the efficiency as high as 100%.THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF

42、BASIC MEDICAL SCIENCESGenomic editing test in pig parthenogenetic embryosTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESGeneration of gene-modified pigsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESEfficient in vivo and ex vivo somatic precise genomic editing i

43、n mice via CRISPR-Cas9THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESPaper1: CRISPR-Cas9 Knockin Micefor Genome Editing and Cancer Modeling (Cell, 2014, 159: 440)pThe objective of this workTo establish a mouse model to facilitate simultaneous multiple gene modification in vivoTHIR

44、D MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpStrategy of this workInducible or constitutive Cas9 expressing miceEx vivo genomic editing by deliver sgRNA vectorsIn vivo genomic editing by deliver sgRNA vectorsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpConstru

45、ction of Cas9 knock-in miceLSL: Loxp-Stop-Loxp; P2A: protein self-cleavage siteTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESCharacterization of Cas9 knock-in micepCharacterization of Cas9/knock-in miceData from constitutive Cas9-expressing mice (Cas9 knock-in X beta-actin-Cre)TH

46、IRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESData from inducible Cas9-expressing mice (Cas9 knock-in X TH-IRES-Cre mice)THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpEx vivo genomic edition in primary Dendritic CellsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF

47、BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESEx vivo genetic editing in brainTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESIn vivo modeling multigenic cancer mutationsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTHIR

48、D MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESLung tumor derived from multigenic cancer mutationsTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpLarge animals can be better models?Minipig?Monkeys?THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESPap

49、er 2: Rapid modeling of cooperating genetic events incancer through somatic genome editing (Nature, 2014, doi:10.1038/nature13906)Somatic mutations have been recognized as causing events for cancer developmentAn effective and convenient in vivo approach to functionally characterize oncogenes will su

50、rely greatly facilitate cancer researchesConventional in vivo genomic editing technology is labor-intensive and highly inefficient.THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpDesign of this workKrasLSL-G12D/+KrasLSL-G12D/+;p53fl/fl THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF B

51、ASIC MEDICAL SCIENCESpResults1: Pathology of tumors derived from Cas9/sgRNA-mediated somatic genetic edition THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpResults 2: Histopathological characterization of tumoursTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpR

52、esults 3: mutation distribution in target siteTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESEfficient Ablation of Genes in Human HematopoieticStem and Effector Cells using CRISPR/Cas9(-Cell Stem Cell 2014, 15, 643652)THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIEN

53、CES第二节第二节 利用正向遗传学研究基因利用正向遗传学研究基因功能功能THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES一、化学诱变一、化学诱变THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES诱变诱变-引向重大科学发现的途径引向重大科学发现的途径THIRD MILITARY MEDICAL UNIV

54、ERSITYCOLLEGE OF BASIC MEDICAL SCIENCESX射线的应用射线的应用pX射线的应用：果蝇 斑马鱼（1989年开始）pX射线的缺点：大段染色体的缺失，导致多个基因同时缺失，难以确定真正引起突变表型的基因THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU的发现的发现p1994 年，德国Tebingen 研究所的Nsslein-Volhard 小组花了2年时间用ENU（N-ethyl-N-nitrosourea, N-乙基-N-亚硝基脲)对斑马鱼进行饱和诱变筛选 。THIRD MI

55、LITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU诱变特点诱变特点-1p ENU 是一种化学诱变剂, 它通过对基因组DNA 碱基的烷基化修饰，诱导DNA 在复制时发生错配而产生单个碱基的突变p 目前已经是公认的最强的小鼠诱变剂p 雄鼠生殖细胞的突变率及后代的数量远远大于雌鼠 p ENU进入雄鼠体内，导致精原细胞大量死亡，剩余精原细胞通过分裂重建睾丸结构，ENU不借助任何代谢过程，直接渗入精原细胞的细胞核与碱基结合，影响生殖细胞的DNA复制，主要导致精子的碱基发生点突变，突变的种类包括碱基间的颠换、转换或移码突变。THIRD

56、 MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU诱变特点诱变特点-2p突变率pSpecific locus mutation rate of 1 in 1,000 gametes. Every 1,000 mice carry a new ENU hit at any locusp诱变结果和人遗传疾病相似诱变结果和人遗传疾病相似pnull allelesppartial loss-of-functionppartial gain-of-functionTHIRD MILITARY MEDICAL UNIVERSIT

57、YCOLLEGE OF BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU mouse mutagenesis projectsp German Research Center for environment and Health (GmbH, formally the GSF), Germany,1997p Medical Rese

58、arch Council (mrC) , UK,1997p RIKEN, Japan,1999p Australian National University(Anu), Australia, 1999p Many more ENU mouse mutagenesis projects were funded, 2000p Approximately twenty such projects have been organized, each of which set itself the primary goal to produce and analyse the phenotypes o

59、f10,000 G1 miceTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES ENU人工诱变已经在果蝇、斑马鱼、小鼠等动物中被证明是一种发现新的功能基因的有效方法，但在大动物上进行人工诱变以创造新的基因资源在国际上仍属空白。小鼠小鼠ENUENU诱变产生的突变性状诱变产生的突变性状THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES小鼠小鼠ENU诱变发现新基因的标志性成果诱变发现新基因的标志性成果p家族性多发性腺癌（human fa

60、milial adenomatous polyposis）pScience. 1990. 247(4940): 322-4.pScience. 1992. 256(5057): 668-70.p生物节律基因（clock）pScience. 1994. 264(5159): 719-25.pCell. 1997. 89(4): 641-53. THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU诱变技术的发展：结合基因驱动，建立诱变技术的发展：结合基因驱动，建立任意基因的突变模型任意基因的突变模型THIRD M