系统信号通路模型课件.pptVIP

Pathway Profiling System Contents Introduction 1 2 Associated vectors 3 experiment 4 Signal transduction research The Pathway Profiling Systems provide a means to assess signal transduction pathway activation?in vivo These systems consist of sets of vectors that each contain a distinct cis-acting enhancer element upstream of a reporter gene 1. Introduction Broad-spectrum profiling ： SEAP （分泌型碱性磷酸酶） System or Luciferase（荧光素酶）Systems SEAP2 Luciferase5 Luciferase 3t Luciferase2 Pathway profiling system More details Luciferase4 2.Signal transduction research Luciferase4 Luciferase2 Luciferase3 SEAP2 investigate nuclear hormone receptors studies of PKC calcium signaling pathways studies of cell proliferation differentiation studies of checkpoints in the cell cycle function Luciferase5 studies of cell inflammation proliferation 2.1 报告基因 secreted alkaline phosphatase reporter （SEAP，分泌型碱性磷酸酶报告基因）： 是人胎盘碱性磷酸酶突交体，具有耐热性，能分泌到细胞外，而且在L-高精氨酸(L-homoarginine)存在下活性亦不变（内源性的碱性磷酸酶会在其存在下失活）。 --由于该酶能分泌到细胞外，在检测时，可以在不破坏细胞的情况下，任意时间都可以取细胞培养上清液进行重复的，动态的检测，且被检测细胞还可以用做其它的用途。是一个很有前途的报告基因。 以间硝基苯磷酸盐(PNPP)为底物时可用标准的比色法测定酶活性，操作简单，反应时间短，价格廉价，但灵敏度低。以黄素腺嘌呤二核苷酸磷酸为底物进行比色测定，其灵敏度增高。 SEAP可催化D-荧光素-O-磷酸盐水解生成D-荧光素，后者又可作为荧光素酶的底物，此即两步生物发光法检测酶活性的原理。此方法灵敏度高，接近于荧光素酶报告基因的检测，还可用一步化学发光法检测酶活性。 Luciferase reportor(荧光素酶报告基因): 荧光素酶(luciferase)催化底物荧光素的转化，发射出光子。 目前较灵敏的检测系统为双荧光素酶检测系统萤火虫荧光素酶(Firefly luciferase)和海肾荧光素酶(Ranilla luciferase)催化的发光反应。 2.2 3.Associated vectors control Generalized map of the pathway profiling vectors The cis-acting enhancer element vector constructs in the pathway profiling systems pTAL-SEAP/Luc vectors can be used to determine the background signals associated with the culture medium.  Aditionally, these vectors can be used for studying putative enhancer elements, which can be cloned into the MCS. Map of pTAL-SEAP/Luc pSEAP2-Control constiutively expresses SEAP in most cell types, which makes it ieal for estabilshing transfection