基因定点整合.ppt

Gene Targeting GT是指在基因组的原位实现对基因的定点突变、定点整合、基因置换及基因修复等。对基因功能的研究提供了极大的方便，比基因的反义抑制和RNAi更直观可靠，尤其在鉴定基因家族成员的功能时更是如此。 Gene targeting (GT) refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome by homologous recombination (HR) and, often, to the conversion of the endogenous gene into a designed sequence, i.e. base changes or gene disruptions through gene replacements. 同源重组介导的GT在细菌、真菌、低等藻类和动物中已成功广泛的应用，而在高等植物的应用研究进展缓慢，重组率很低（1%），仅为非同源重组的万分之一。 GT在植物中的进展 Paszkowski等1988年首次报道在烟草中作的基因打靶的尝试。 Kammerer和Cove(1996)首次在小立碗藓中实现了基因的同源重组,打靶效率达到90％,近似于酵母的95％ 。 Terada 等（2002；2007）利用高效正负向筛选标记筛选同源重组子的策略,白喉毒素基因DT-A作负向筛选标记,成功的在水稻Waxy或Adh2中整合进一个潮霉素抗性基因htp，实现对靶基因的敲除。打靶效率达到1％。 打靶的效率与DNA断裂双链的修复过程中倾向于同源重组还是非同源重组有很大的关系。 提高打靶效率的方法 通过表达内源或外源同源重组相关的酶提高同源重组率,如RAD、RecQ和RecA等。 引入重组酶体系Cre/lox。 锌指核酶ZFNs（zinc finger nucleases）引入定点双链断裂。ZFN由一个DNA识别域和一个非特异性核酸内切酶构成,具有很强的特异性和可塑性。 Schematic of the target reporter and zinc finger recognition sequences. a Target reporter construct with tandem, overlapping, partial gfp gene fragments, a 3′ pat selectable marker gene fragment, left and right homologous sequences and zinc finger binding sites. b Recognition sequences for ZFN-1 and ZFN-2. Structural features of the zinc finger nuclease (ZFN) assembly and expression vector systems.A zinc finger protein (ZFP) coding sequence can be assembled by Klenow/PCR using a combination of overlapping backbone and sequence-dependent oligonucleotides fused to the FokI endonuclease domain in pSAT4.35SP.NLS-FokI, producing the plant expression vector pSAT4.35SP.ZFN. The entire ZFN coding sequence can be transferred onto a pET28.XH-based vector producing a pET28.XH-ZFN vector, suitable for ZFN expression in bacterial cells and for in vivo digestion assays. The pSAT4.35SP.ZFN can be modified by replacing the 35S constitutive promoter with a heat-shock-inducible promoter, producing a plasmid that is useful for the whole-plant DNA repair assay. A plant-selectable marker, a ZFN and a