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1、Transgene and gene knockout in studying gene functions,1,功能缺失(Loss of function)： KO, CKO, RNAi获得功能(Gain of function)：Transgenic, virus-based 在体 in vivo与离体 in vitro描述性报告 vs 机理研究报告 研究设计：假说(可能性)验证总结(论文),研究基因功能的两个策略,2,小鼠基因敲除和转基因,这两项技术是用来制备目的基因失活和表达的小鼠。优势：在体研究基因的功能。应用和意义：使得遗传学、肿瘤学、免疫学、发育生物学等学科取得了惊人的进步，这些
2、研究潜在的商业性价值不可估量。,3,基因敲除（knock out ）和基因敲入（ knock in） KO过程也称基因打靶(gene targeting)，利用DNA同源重组，在基因组中的某一特定部位进行定点的基因置换，用无功能基因替换目的基因或Delete目的基因。 根据需要（如验证A与B在功能上有某种关联），在敲除基因A时，将有功能的基因B放在A基因的位置，即A是KO，B是Knock in。,4,基因敲除的基本程序,1 、构建打靶载体 Targeting vector 1）同源序列 Homologous arms 2）打靶载体常含两种筛选标志,5,ositive selectionneor
3、 ：新霉素抗性基因，neor基因存在于打靶载体内，当重组（同源或随机整合）后，ES细胞能在含新霉素G418的培养液中生长negative selection1. HSV-tk：单纯疱疹病毒的胸腺嘧啶激酶基因，该基因产物可分解单核苷酸类似物(ganciclovir)生成毒性产物，产生自杀效果 同源重组时：Tk-基因丢失，ES细胞存活随机整合时：Tk-基因存在，ES细胞死亡2. DTA (diphtheria fragment A) : 不用给药,6,2、将载体导入ES细胞,Targeting vector introduced by electroporation,Targeting gene,
4、7,Homologous recombination,8,Recombinants with random insertion-containing both neo and tk,Selection for neor positive,Selection for TK negative,ES cells with homologous recombination,ES Cells,3. 筛选进行了同源重组的ES细胞,9,4 、将基因敲除ES细胞注射入囊胚，形成嵌合胚胎，得到嵌合体小鼠，而后筛选培育阳性子代小鼠。,10,Conventional Knock out,11,Mutation of
5、 Lmx1b in mice,In forelimb, dorsal feature (hair follicles) is replaced by ventral one (footpads).,(Chen et al., Nat. Genetics, 1998),12,Lmx1b is essential for development of 5-HT+ neurons,(Ding et al., Nat. Neurosci., 2003),13,Conditional knock out (CKO)(Tissue- or cell type-specific),条件Floxed mice
6、目的基因（A）被两个LoxP位点包绕Cre mice转基因小鼠，Cre在特定细胞内表达同时表达目的基因（A）和Cre的细胞内的A基因被敲除，其他细胞表达的A基因不受影响,14,Raphe nuclei-specific deletion of Lmx1b,(Dai et al., PNAS, 2008),(functional Cre examination with Rosa26 reporter mice ),15,Mating,viable,16,CKO mice lose essentially all 5-HTergic neurons in the brain,17,Time-co
7、ntrol CKO,(Dai et al., J Comp Neurol, 2008),18,Generation of Pet1-CreER mice,(Song et al., PlosOne),CreER位于胞浆内，不具有活性；在tamoxifen存在情况下，转位入核发挥作用。,19,Lmx1b is required for 5-HT expression in Adult Brain,5-HT immunostaining,tph2 immunostaining,20,Other methods in gene Knock out,ENU-based mutagenesis N-et
8、hyl-N-nitrosourea (ENU) was developed as an efficient mutagen that would cause point mutations and small deletions in mice. It easy to design closely spaced DNA markers for mapping of mutations.,21,The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR): RNA-Guided Endonuclease techno
9、logy for genome engineering. Two components: (1) a guide RNA (2) an endonuclease, CRISPR associated (Cas) nuclease, Cas9.,http:/www.addgene.org/CRISPR/guide/,22,Transgene,23,Transgene,Principles: 1. which type of cells you are interested(e.g. islet cells, dopaminergic neurons)2. what gene you want t
10、o express Making vectors1. Known promotor and enhancer2. Unknown cases BAC-based method,24,Founder mice:transgenic mice that develop from the injected eggsTransgenic mice:mice that are determined to presence of the injected geneStable line:due to multiple insertion,25,美国The Jackson Laboratory世界最大的小鼠
11、品系仓库,26,南京国家小鼠资源中心,27,资源库坐落于南京市浦口高新区，占地100亩，建筑面积7800平方米，小鼠笼位3万多个，各项指标已通过省实验动物环境与设施的监测。引进和自主构建小鼠品系达150多种。,南京国家小鼠资源中心,28,SPF饲养要求,Specific pathogen free-SPF饲养环境和动物没有特定的病原感染动物福利：环境、对待动物的方式使用量：最小化为原则,29,Mouse Care: Barrier Accommodation with Individually Ventilated Cage Racks,空气过滤（万级）、控温和控湿动物防护：更衣、口罩、手套、
12、头套,30,子宫内胚胎电转技术in utero electroporation,31,功能缺失（LOF）： KO, CKO, RNAi获得功能（GOF）：Transgenic, virus-based 不利的因素:Labor- and time-consumingSpecialist and equipment are needed,研究分子功能的两个策略,32,在体电转技术，可以在任何组织，任何时期（发育和成年）进行LOF和GOF实验LOF：RNAi (virus-based, or vector-based)GOF: Over-expressing what you want依赖于稳定可靠
13、的在体电转仪的出现,33,In vivo 电转的原理,电转后瞬间细胞膜上被击出的孔,带电的质粒在电场作用下胞内的形式,34,Vectors,LOF (RNAi)：pSuperGOF: pCAGGS，强大启动子，表达时间长,35,子宫内胚胎电转In Utero Electroporation,动物: 鸡胚，小鼠，大鼠胎龄：鸡胚：越小越容易小鼠、大鼠：越大越容易部位：神经系统、肌肉、胃肠道、皮肤、心脏 肾脏、眼睛等等,36,实例一 神经细胞迁移,Ding YQ et al., Development, 2005,37,Misexpression of DCC induces ventral mig
14、ration of spinal neurons,Ding YQ et al., Development, 2005,38,Ding YQ et al., Development, 2004,实例二,39,Knock down-RNAi,RNAi reveals that doublecortin is required for radial migration in rat neocortex. Nat Neurosci. 2003 6:1277-83,实例三,40,如何开始?,几个原则:在水或中性的缓冲液内质粒是带负电的质粒在电场的作用下可以进入细胞内质粒必须在目的细胞的周围BTX has
15、 a product (ECM 830) that is able to do this job efficiently (http:/),41,Misexpression vector: pCAGGGSRNAi vector: pSuperInjection vectors into brain ventricleElectric pulsesPut embryos backFor detailed procedures, please read the paper in Dev Bio, 240:237, 2003, by T Saito.,42,电转后GFP在大脑皮层的表达,Saito
16、& Nakatsuji., Dev Bio, 2001,43,研究大脑皮层神经元产生迁移,Temporal and positional changes of electroporated neurons in the cortex,(Chen et al., Nat Neurosci, 2008),44,RNAi 方法降低Dcx 在大脑皮层的表达导致皮层板层形成障碍,(Bai J et al., Nature Neurosci, 2003),Mutations in DCX in humans cause malformation of the cerebral neocortex. Par
17、adoxically, genetic deletion of Dcx in mice does not cause neocortical malformation. However,. Technically, electroporation alone is enough for.,45,其他脑区,Cingulate cortexHippocampusStriatumThalamus HypothalamusBrain stemDRG,46,胚胎电转注意事项之一：时间点选择,利用神经系统的细胞产生的时间不同，可选择转入至目的细胞 For neocortex:Layers V-VI: E1
18、2.5Layer IV: E13.5-E14.5Layers II-III: E15.5注意：应考虑到对神经母细胞的影响,47,在成年神经系统内的应用,定位注射质粒到目的脑区：电击：电压、 Pulse数 观察改变：细胞水平、行为学,（Wei F et al., J Neurosci, 2002）,48,更多应用 一次电转多种质粒,Green-EYFP; Blue-ECFP; Red-DsRed,49,Use Tet-on or Tet-off system to temporally control expression of genes of interest. Use Cre-Loxp s
19、ystem to transiently express genes of interest.,(Mizutami & Saito, Development, 2005),适时表达，适时中止,50,鸡胚电转,More widely usedEasy to accessEggs are cheapfor both GOF and LOF,51,(Odani et al., Development, Growth and Differentiation, 200, Vol 6),52,53,(Xie et al., Nat Cell Biol, 2005),Over-express dominan
20、t-negative protein,54,Knock down of gene expression causes abnormal cell death,55,Misexpression of three genes induces ectopically expression of 5-HT in spinal cord,(Cheng L et al., J Neurosci., 2003),56,鸡胚心脏内过度表达基因,57,成年小鼠肌肉内过度表达基因,(Miyazaki et al., Development, Growth and Differentiation, 2008, Vo
21、l 6),58,小鼠视网膜内过度表达基因,59,(Zheng MH, et al., Mol Brain, 2009),60,小鼠肾脏内过度表达基因,61,大鼠睾丸电转,(Yomgogida, Development, Growth and Differentiation, 200, Vol 6),62,电转mRNA至 Xenopus,63,体外培养组织,没有任何技术问题脑片电转后培养单细胞局部少量细胞,(Uesaka et al., Development, Growth and Differentiation, 2008, Vol 6),64,Summary I,Explore possi
22、ble function of a new gene/protein during development as well as in adult优点:Easy to perform in a lab Time- and labor-savingNot expensive to set up,65,Disadvantages:Only a small number of neurons are electroporatedAn attention should be paid to possible impairment of progenitor cells, when interested genes are not expressed in the progenitor cells Electroporation itself may cause cell death, please keep in mindOther approaches should be taken to address your question,Summary II,66,谢 谢,http:/ Growth and Differentiation,67,
