核心課程遺傳工程與悉生動物模型

1、THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES遺傳工程與悉生動物模型遺傳工程與悉生動物模型Genetic Manipulated and Human flora-associated Genetic Manipulated and Human flora-associated (HFA) Animal Models(HFA) Animal Models 生命科學核心課程王 勇第三軍醫(yī)大學基礎部實驗動物學教研室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?疾病動物模型研究需求針對生命科學關鍵問題疾病動物模型無菌技術遺傳工程技術解析機體自身(宿主)基因組功能解析機體寄生菌群(宏)基因組能生長發(fā)育健康疾病與菌群關系密切Science.2012;336THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES第一章第一章 遺傳工程動物遺傳工程動物Genetically manipulated animalsTHIRD MILITARY MEDIC

4、AL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES利用模式動物研究基因與疾病關系的兩利用模式動物研究基因與疾病關系的兩種模式種模式THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp反向遺傳學反向遺傳學（reverse genetics）：從基因到表型：從從特定基因的改造特定基因的改造到到整體動物表型整體動物表型分析：分析：通過向動物基因組增加一個基因（轉基因）或通過向動物基因組增加一個基因（轉基因）或刪除一個基因（基因敲出），觀察動物表型變化刪除一個基因（基因敲出），觀

5、察動物表型變化，進而分析基因與表型的關系，研究基因功能，進而分析基因與表型的關系，研究基因功能THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp優(yōu)點：能深入、系統地研究特定基因功能，能在較短能深入、系統地研究特定基因功能，能在較短時間獲得詳實的研究資料時間獲得詳實的研究資料p缺點：動物表型往往受多基因調控，一個基因改變不動物表型往往受多基因調控，一個基因改變不能反應生物學機制的全貌能反應生物學機制的全貌單純的表型分析往往會單純的表型分析往往會“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正向遺傳學(Forward Genetics)：從表型到基因 從特定表型改變到基因分析：通過高效率的、高通量的通過高效率的、高通量的隨機誘變技術隨機誘變技術大規(guī)模大規(guī)模制備突變突變動物，然后從大量的基因突變動制備突變突變動物，然后從大量的基因突變動物中篩選物中篩選表型變異個體表型變異個體，最后通過遺傳學分析，最后通過遺傳學分析、染色體定位、分子克隆等手段，最終克隆、染色體定位、分子克隆等手段，最終克隆導導致表型變異的突變基因致表型變異的突變基因THIRD MILITARY MEDICAL UNI

7、VERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp優(yōu)點：優(yōu)點：可獲得較完整的遺傳信息可獲得較完整的遺傳信息p缺點：缺點：遺傳分析與相關基因克隆是一繁瑣過程遺傳分析與相關基因克隆是一繁瑣過程需要大規(guī)模飼養(yǎng)動物，成本高需要大規(guī)模飼養(yǎng)動物，成本高THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES第一節(jié)第一節(jié) 利用反向遺傳學模式研究利用反向遺傳學模式研究基因功能基因功能THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCI

8、ENCES利用正向遺傳學研究基因功能的基本思路利用正向遺傳學研究基因功能的基本思路文獻查閱、課題設計確定候選基因RNA導向的核酶技術(CRISPR/Cas9)基于DNA同源重組的基因敲出基于位點特異性核酶(ZFN,TALEN)的基因敲出基因敲出（knock-out)動物模型系統、綜合的表型分析正?；蜣D入基因回復突變動物模型確定基因功能THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES一、基因敲出動物一、基因敲出動物(Gene knock-out animal)THIRD MILITARY MEDICAL UNI

9、VERSITYCOLLEGE OF BASIC MEDICAL SCIENCES候選基因確定候選基因確定p依據課題目標，解析課題目標所涉及的生物學過程p梳理生物學過程中所涉及的可能信號通路（網絡）或分子機制p選擇關鍵節(jié)點中的關鍵基因THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES全球性的規(guī)?；蚯贸鲇媱澣蛐缘囊?guī)?；蚯贸鲇媱?initiative to knockout every geneTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL

10、SCIENCESHow to knock out a gene？在Evan等教授建立胚胎干細胞的基礎上，上世紀80年代由Capecchi, Smithies 等建立了基于胚胎干細胞(ES細胞)DNA同源重組的基因敲除技術 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細胞中通過細胞中通過DNA同源重組同源重組，將已發(fā)，將已發(fā)生失活突變的基

11、因打靶載體置換基因組生失活突變的基因打靶載體置換基因組內野生型的目標基因位點，使目標基因內野生型的目標基因位點，使目標基因活性消失活性消失 Oliver Smithies and Mario R. CapecchiTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES外顯子外顯子785687Neo5TKNeo敲除載體野生拷貝基因敲除拷貝：基因敲除拷貝：失去正常結構或功能失去正常結構或功能同源重組基因敲除的原理圖基因敲除的原理圖578THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF

12、 BASIC MEDICAL SCIENCES雙向篩選獲得基因敲出的雙向篩選獲得基因敲出的ES細胞細胞1234TK2NeoNeoG418 抗性GANC抗性1.1.同源重組同源重組2TKNeo1234NeoTKNeo2214G418 抗性GANC敏感2.隨機整合3.沒整合G418 敏感GANC抗性THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpStep2將發(fā)生將發(fā)生同源重組的同源重組的ES細胞輸入囊胚，建立細胞輸入囊胚，建立堪合體胚胎，進而獲得堪合體動物?？昂象w胚胎，進而獲得堪合體動物。 Martin J. E

13、vans THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESpStep3讓堪合體動物與野生型動物的交配，通讓堪合體動物與野生型動物的交配，通過其過其源自外源源自外源ES細胞的配子細胞的配子獲得基因敲獲得基因敲出的雜合體動物，進而獲得純合體動物出的雜合體動物，進而獲得純合體動物THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESStep1Step2Step3Summary of this technologyTHIRD MILITARY

14、 MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESCharacteristics of knockout(KO) animalsp KO小鼠的所有細胞，包括生殖細胞，都缺乏同一特異基因表達p KO動物可同時從DNA、RNA、蛋白質、細胞、組織、器官、整體動物等多種水平直接，動態(tài)地觀察基因在整體動物內的活動情況以及改變該基因后引起的表型變化.p 若靶基因為發(fā)育或生長的必須基因，往往會導致動物發(fā)育異?；蜻^早死亡THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESCo

15、nditional knock-outp在特定的細胞、組織或器官中特異性敲出靶基因，而在其他細胞、組織或器官中靶基因未發(fā)生功能缺失p是目前克服常規(guī)基因敲出技術缺陷的主要手段THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESp位點特異性重組酶-條件性基因敲出的必備工具Cre: Create recombination enzyme在噬菌體中發(fā)現，可引起長36 bp的特殊順序（loxp， location of cross) 間的重組，從而敲除 loxP 間的DNA。 Gu Hua于1995 年首先將其應用在小鼠基因

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-適用于不同物種的基因敲出技術適用于不同物種的基因敲出技術pClassic gene

18、 knock-out technology:高度依賴于永生化、全能性的胚胎干細胞高度依賴于永生化、全能性的胚胎干細胞流程繁雜、周期長，不適用于時代間隔長流程繁雜、周期長，不適用于時代間隔長的大動物的大動物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,鋅指核酶是人工構建的序列特異性核酸內切酶，由兩部分組是人工構建的序列特異性核酸內切酶，由兩部分組成：具備高度序列特異性的成：具備高度序列特異性的DNA結合功能域結合功能域和不具和不具備序列特異性的備序列特異

20、性的DNA內切酶功能域內切酶功能域其中，其中，DNA結合功能域由結合功能域由3-4個鋅指結構域個鋅指結構域組成，每組成，每個鋅指結果識別個鋅指結果識別3bp，則一個鋅指核酶識別，則一個鋅指核酶識別9-12bp一個一個ZFN分子指切斷一條分子指切斷一條DNA鏈鏈THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESZFN介導介導gene knock-out的原理的原理p 由兩個互補的ZFN分子（左向ZFN分子ZFN-L、右向ZFN分子ZFN-R）同時特異性地與靶位點結合，在靶位點導入雙鏈斷裂切口（Double stra

21、nd break，DSB）p 由于DSB的存在啟動了細胞基因組修復機制，由此可實現多種位點特異性遺傳修飾： 細胞通過錯配率很高的（細胞通過錯配率很高的（error-prone）“非同源性末非同源性末端連接端連接”（Non-homologous End Joining,NHEJ）機）機制修復制修復DSB，在，在ZFN靶位點造成靶位點造成堿基插入或缺失堿基插入或缺失，形成，形成位點特異性突變，導致靶基因功能缺失位點特異性突變，導致靶基因功能缺失 若在修復若在修復DSB時胞內存在與靶位點同源的時胞內存在與靶位點同源的DNA片段，則片段，則細胞主要通過細胞主要通過DNA同源重組（同源重組（Homolo

22、gous Recombination, HR）的機制修復的機制修復DSB，由此可實現靶，由此可實現靶基因置換或校正（基因置換或校正（gene replacement or correction）以及外源以及外源DNA片段定點敲入（片段定點敲入（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技術的關鍵技術的關鍵p篩選最佳的鋅指結構域組合（Zinc Finger Arrays）是形成特異性高、親和力強的DNA結合功能域的關鍵p不是每一個依照靶序列組合的Zinc Finger Array都有活性，由此限制了ZFN技術適用性THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCEST

26、ALEN technologypA more powerful genomic editing tool, which is moving over ZFN理論上，針對任何基因的任何序列均可構建有理論上，針對任何基因的任何序列均可構建有活性的活性的TALENTALEN分子分子脫靶效應低于脫靶效應低于ZFNZFN，細胞毒性較弱，細胞毒性較弱目前正作為一種常規(guī)的基因修飾技術在迅速推目前正作為一種常規(guī)的基因修飾技術在迅速推廣和應用，大有替代廣和應用，大有替代ZFNZFN和和RNAiRNAi技術之勢技術之勢THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASI

27、C MEDICAL SCIENCESThe TALEN working mechanismp TALE（Transcription activator -like effectors）是一種源于植物致病菌Xanthomonas的轉錄激活因子，用于在細菌感染宿主細胞時啟動宿主基因表達、以促進細菌感染p 研究發(fā)現，TALE因子與宿主基因靶位點的結合是通過一段模塊重復序列（Modular repeats array)實現的，一個模塊識別一個堿基，模塊與堿基之間呈一一對應的關系p 每個模塊重復序列約34個氨基酸，模塊之間除了第12和13位氨基酸不一樣以外，其余氨基酸序列幾乎一致；而這兩個不一致的兩個氨

28、基酸決定了模塊識別堿基的特異性，被稱為RVD（repeat variable di-residues)THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTALETALE特異性識別堿基特異性識別堿基的原理：的原理：TALEs由數10-30個特異性識別DNA堿基的串聯“重復模塊”和兩側的N-末端及C-末端序列組成（如上圖a）。每個“重復模塊”包含34個氨基酸，第12和13位殘基是靶向識別的關鍵位點，被稱作重復可變的di-residues（RVDs）位點。TALEs上的四種RVDs（如圖b）分別識別四種堿基。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的特異性打靶的原理（同的特異性打靶的原理（同ZFNZFN）：）：將特異性識別靶基因的TALE蛋白與ForKI內切酶的核酶功能域相融合，構建成融合分子TALEN(TALE-nuclease，如上圖); 將

30、2個可與靶位點上下游互補鏈特異性結合的TALEN分子同時結合在靶基因位點，每個TALEN分子切斷一條DNA鏈，兩個TALEN則可在靶基因位點形成DNA雙鏈斷裂（double-strand break，DSB），引入的DSB可激活DNA自我修復，從而引起基因的突變和促進靶位點DNA同源重組。THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTALEN基因敲出原理THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTALEN TALEN

31、技術的發(fā)展歷程技術的發(fā)展歷程THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES利用利用TALEN技術研制的基因敲出動物技術研制的基因敲出動物Science, 2011,29:695THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESNature Biotechnology, 2013,31:23THIRD

32、 MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES首次報道了利用TALEN技術創(chuàng)制了基因敲出大動物（豬、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第二節(jié)第二節(jié) 利用正向遺傳學研究基因利用正向遺傳學研究基因功能功能THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES一、化學誘變一、化學誘變THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESTHIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES誘變誘變-引向重大科學發(fā)現的途徑引向重大科學發(fā)現的途徑THIRD MILITARY MEDICAL UNIV

54、ERSITYCOLLEGE OF BASIC MEDICAL SCIENCESX射線的應用射線的應用pX射線的應用：果蠅 斑馬魚（1989年開始）pX射線的缺點：大段染色體的缺失，導致多個基因同時缺失，難以確定真正引起突變表型的基因THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU的發(fā)現的發(fā)現p1994 年，德國Tebingen 研究所的Nsslein-Volhard 小組花了2年時間用ENU（N-ethyl-N-nitrosourea, N-乙基-N-亞硝基脲)對斑馬魚進行飽和誘變篩選 。THIRD MI

55、LITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU誘變特點誘變特點-1p ENU 是一種化學誘變劑, 它通過對基因組DNA 堿基的烷基化修飾，誘導DNA 在復制時發(fā)生錯配而產生單個堿基的突變p 目前已經是公認的最強的小鼠誘變劑p 雄鼠生殖細胞的突變率及后代的數量遠遠大于雌鼠 p ENU進入雄鼠體內，導致精原細胞大量死亡，剩余精原細胞通過分裂重建睪丸結構，ENU不借助任何代謝過程，直接滲入精原細胞的細胞核與堿基結合，影響生殖細胞的DNA復制，主要導致精子的堿基發(fā)生點突變，突變的種類包括堿基間的顛換、轉換或移碼突變。THIRD

56、 MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU誘變特點誘變特點-2p突變率pSpecific locus mutation rate of 1 in 1,000 gametes. Every 1,000 mice carry a new ENU hit at any locusp誘變結果和人遺傳疾病相似誘變結果和人遺傳疾病相似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人工誘變已經在果蠅、斑馬魚、小鼠等動物中被證明是一種發(fā)現新的功能基因的有效方法，但在大動物上進行人工誘變以創(chuàng)造新的基因資源在國際上仍屬空白。小鼠小鼠ENUENU誘變產生的突變性狀誘變產生的突變性狀THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCES小鼠小鼠ENU誘變發(fā)現新基因的標志性成果誘變發(fā)現新基因的標志性成果p家族性多發(fā)性腺癌（human fa

60、milial adenomatous polyposis）pScience. 1990. 247(4940): 322-4.pScience. 1992. 256(5057): 668-70.p生物節(jié)律基因（clock）pScience. 1994. 264(5159): 719-25.pCell. 1997. 89(4): 641-53. THIRD MILITARY MEDICAL UNIVERSITYCOLLEGE OF BASIC MEDICAL SCIENCESENU誘變技術的發(fā)展：結合基因驅動，建立誘變技術的發(fā)展：結合基因驅動，建立任意基因的突變模型任意基因的突變模型THIRD M