《系統(tǒng)信號通路模型》ppt課件

1、Pathway Profiling SystemContentsIntroduction12Associated vectors3experiment4Signal 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 el

2、ement upstream of a reporter gene1. IntroductionBroad-spectrum profiling ： SEAP 分泌型堿性磷酸酶 System or Luciferase熒光素酶SystemsSEAP2Luciferase5 Luciferase 3tLuciferase2Pathway profiling systemMore detailsLuciferase42.Signal transduction researchLuciferase4Luciferase2Luciferase3SEAP2investigate nuclear horm

3、one receptors studies of PKC & calcium signaling pathways studies of cell proliferation & differentiationstudies of checkpoints in the cell cycle functionLuciferase5studies of cell inflammation & proliferation 2.1 報告基因 secreted alkaline phosphatase reporter SEAP，分泌型堿性磷酸酶報告基因： 是人胎盤堿性磷酸酶突交

4、體，具有耐熱性，能分泌到細胞外，而且在L-高精氨酸(L-homoarginine)存在下活性亦不變內(nèi)源性的堿性磷酸酶會在其存在下失活。 -由于該酶能分泌到細胞外，在檢測時，可以在不破壞細胞的情況下，恣意時間都可以取細胞培育上清液進展反復(fù)的，動態(tài)的檢測，且被檢測細胞還可以用做其它的用途。是一個很有出路的報告基因。 以間硝基苯磷酸鹽(PNPP)為底物時可用規(guī)范的比色法測定酶活性，操作簡單，反響時間短，價錢廉價，但靈敏度低。以黃素腺嘌呤二核苷酸磷酸為底物進展比色測定，其靈敏度增高。 SEAP可催化D-熒光素-O-磷酸鹽水解生成D-熒光素，后者又可作為熒光素酶的底物，此即兩步生物發(fā)光法檢測酶活性的原理

5、。此方法靈敏度高，接近于熒光素酶報告基因的檢測，還可用一步化學(xué)發(fā)光法檢測酶活性。 Luciferase reportor(熒光素酶報告基因): 熒光素酶(luciferase)催化底物熒光素的轉(zhuǎn)化，發(fā)射出光子。 目前較靈敏的檢測系統(tǒng)為雙熒光素酶檢測系統(tǒng)螢火蟲熒光素酶(Firefly luciferase)和海腎熒光素酶(Ranilla luciferase)催化的發(fā)光反響。2.23.Associated vectorscontrolGeneralized map of the pathway profiling vectorsThe cis-acting enhancer element ve

6、ctor 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-Cont

7、rol constiutively expresses SEAP in most cell types, which makes it ieal for estabilshing transfection efficiency and optimizing your SEAP assay detection method 4.experimentprocedureAfter transfection: it is important to remove serum from the meium to ensure properinduction of th reporter Serum ind

8、uces various signaling pathways causing high background.About stimulus: perform a time course study by collecting samples at various time points.SEAP-can collect samples from a single cell culture Luciferase-must set up multiple cell cultures to collect at different time pointsLow transfection efficiency: poor quality DNA,PH, et al.Cell density- keep constant; 50-80% confluent at the time of transfect