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1、基因敲除,Transgenic vs. “knock-out”,“Knockout”,“Transgenic”,“Transgenic”: an organism that has had DNA introduced into one or more of its cells artificially DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum Random insertion -Often multiple copies,“knockout”:
2、DNA is introduced first into embryonic stem (ES) cells. ES cells that have undergone homologous recombination are identified and injected into a 4 day old mouse embryo - a blastocyst targeted insertion - single copy,Transgenic production,Transgenic mice are often generated to 1. characterize the abi
3、lity of a promoter to direct tissue-specific gene expression e.g. a promoter can be attached to a reporter gene such as LacZ (Beta-galactosidase) or GFP (Green Fluorescent Protein) 2. examine the effects of over-expressing and mis-expressing endogenous or foreign genes at specific times and location
4、s in the animals,Promoter of gene of interest,cDNA copy of gene of interest,Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene. Neural structures expressing the reporter transgene are dark b
5、lue-green. (Dr. Anne Calof),Tail tip,9.5 day embryos GFP and wt,GFP transgenic mouse,基因敲除主要是在胚胎干细胞(embryonic stem ceils，ESC)水平进行操作。 胚胎干细胞是早期胚胎细胞经体外培养建立的全能细胞系，在体外培养时保持了未分化状态，可以传代增殖， 在发育上类似于早期胚胎细胞团的细胞，具有与早期胚胎细胞相似的分化潜能和正常整倍体核型两大特点，是研究哺乳动物个体发育、胚胎分化以及性状遗传机制的理想模型。 首先在这种细胞内进行分子水平的基因操作，之后再使这种已经发生了基因改变的细胞发育成
6、为一个完整的生物体。 这样就可以在整个生物体内实现预期的基因改变。,基因敲除,Vector design,Recombinant DNA methods: Simple KO Structural gene desired (e.g. Cyp2E1) to be “knocked out“ is replaced partly or completely by a positive selection marker. (knock out function!) Vector DNA to enable the molecules to be inserted into host DNA mol
7、ecules,HSV-TK,Neomycin resistance gene (positive selection),Herpes simplex virus Thymidine Kinase (negative selection),Resistant to neomycin,Sensitive to ganciclovir,Preparation of Embryonic Stem Cells,Harvested from the inner cell mass of mouse blastocysts Grown in culture and retain their full pot
8、ential to produce all the cells of the mature animal, including its gametes,Selecting ES Cells with KO,Injecting fertilized eggs,The eggs are harvested 0.5 dpc (superovulated or natural matings) The DNA is usually injected into the male pronucleus The eggs can be transferred the same day or the next
9、 (2-cell) into pseudo-pregnant female oviducts,Successfully transformed ES cells are injected into blastocysts,Pseudopregnant females and vasectomized males,Female mice can be tricked into thinking they are pregnant A mouse in estrus is mated with a vasectomized male pseudopregnancy If eggs (blastoc
10、ysts) implanted will become truly pregnant and will give birth to live offspring,General procedure for producing gene-targeted knockout mice. ES cells heterozygous for a knockout mutation in a gene of interest and homozygous for a marker gene (e.g. black coat color) are transplanted into the embryos
11、 that are homozygous for an alternate marker (e.g. white coat color). The early embryos then are implanted into a pseudopregnant female. Some of the resulting progeny are chimeras, indicated by their black and white coats. Chimeric mice then are backcrossed to white mice black progeny from this mati
12、ng have ES-derived cells in their germ line. By isolating DNA from tail tissue, its possible to identify black mice heterozygous for the knockout allele. Intercrossing of these black mice produces knockout mice.,Offspring - Chimeric founders,Black mouse - no apparent ES cell contribution,Chimeric fo
13、under - strong ES cell contribution,Chimeric founder - weaker ES cell contribution,germ-line transmission - usually the ES cells are derived from a 129 strain (agouti or white colour) and the ES cells are injected into a C57Bl/6 blastocyst (black). The more that the ES cells contribute to the genome
14、 of the mouse, the more the coat colour will be agouti. The chimera mouse is usually “tiger” striped.,小分子RNA - 生命科学的新宠,人类对生命的探索,垃圾DNA ! Junk DNA ， Genetic Wasteland,Non-coding Sequence,当然不是！ Sure not !,小RNA的发现,Why It Got Lost in the Shuffle for so long,世纪钻石,小分子RNA研究, 摘取2002年度SCIENCE十大发现第一名的桂冠,小RNA的研
15、究为什么沉寂多年？ Why It Got Lost in the Shuffle for so long,1969年，Britten and Davidson 预测：真核细胞 中基因表达的开关与RNA的识别直接联系,2001年前: 德国学者另辟蹊径，专门从事对 “垃圾基因”的研究，提出了“RNA组学的概念”,同时, 加拿大学者Mattick 认为非 编码 RNA是决定基因复杂 调控的关键网络分子,精彩纷呈的世界,miRNA and siRNA,siRNA and miRNA 的发现过程；,作用的方式和差别；,miRNA 的功能研究,1990年：2个研究组报道了正义RNA 具有“共抑制”现象,S
16、iRNA 的发现,Petunia 牵牛花,1998年: 第一次在线虫中证实dsRNA, 在抑制基因表达中的功能,Neg. control Uninjected,Antisense RNA dsRNA,Nature 1998 391:806-811,Mex-3 mRNA detection in embryos by in situ hybridization,RNAi的发现,1995年，Guo S等试图阻断秀丽新小杆线虫（C. elegans）中的par-1基因的表达。 设计： 反义RNA 特异性地阻断par-1基因的表达； 正义RNA 以期观察到基因表达的增强。 结果: 二者都同样地切断了par-1基因的表达途径。这是与传统上对反义RNA技术的解释不相符合。该研究小组一直没能给这个意外以合理解释。,RNAi的提出,直到1998年2月，Fire A和Mello C才首次揭开这个悬疑之谜。 他们将体外转录得到的单链RNA纯化后注射线虫时发现，基因抑制效应变得十分微弱；而经过纯化的双链RNA却正好相反，能够高效特异性阻断相应基因的表达。 他
