转基因治病模型课件

1、Transgenic Mutation Animal Assays 转基因动物致突变模型转基因动物致突变模型 Tao Chen, Ph.D. 陈涛，博士 National Center for Toxicological Research, US FDA 美国食品药品管理局国家毒理研究中心 Outline 概要概要 Introduction 导言导言 What is Mutation? 什么是突变？ Mutation and Cancer 突变和癌症突变和癌症 Mutation as a biomarker of cancer 突变可用作癌症的生物指标突变可用作癌症的生物指标 Transgen

2、ic mutation systems 转基因致突变系统转基因致突变系统 Transgenic animals for mutation assays 检测突变的转基因动物转基因动物 Shuttle vector 穿梭载体 clacZlacIAmpr SL cos cI857cro PRE OPQ A J cos cIIQR PR Transgenic rodent mutation models 转基因动物转基因动物突变模型 Big Blue Rats 大蓝大鼠大蓝大鼠 Isolation of genomic DNA TreatmentMutant manifestation Collec

3、tion of tissues Phage packaging or plasmid recovery Infection of E. coli Plating and plaque formation Titer plate Mutant plate Positive selection Sequencing of mutants Calculation of mutant frequency Color selection lacZ lacI Procedure for transgenic rodent mutation assays 转基因动物致转基因动物致突变实验步骤 Treatme

4、nt 药剂处理, Mutant manifestation time 突变体显示时间突变体显示时间 Collection of tissues 组织收集 Isolation of genomic DNA 分离染色体组DNA Statistics and criteria 统计和准则 Validation of Transgenic Systems 转基因系统的转基因系统的确证确证 MFs in Lymphocyte Hprt and lacI vs. Liver lacI in N-OH-AAF Treated Rats N-OH-AAF引诱的淋巴细胞Hprt和lacI基因突变频率以及肝基因突

5、变频率 MF (x 10-6) Hprt vs. lacI MF Induced by Thiotepa in Lymphocytes Thiotepa引诱的淋巴细胞Hprt和lacI基因突变频率 MF (x 10-6) Comparisons of mutations in the Hprt and lacI genes Hprt和lacI基因突变的比较 Transgenic mouse mutation assay vs. mouse bone marrow micronucleus test for predicting mouse carcinogenicity 小鼠致癌性的预测：转基

6、因小鼠致突变测试对小鼠骨髓微核体测试小鼠致癌性的预测：转基因小鼠致突变测试对小鼠骨髓微核体测试 Summary on results in transgenic rodent assays vs. genotoxicity in vitro tests 致突变结果比较：转基因动物致突变测试对体外遗传毒理测试致突变结果比较：转基因动物致突变测试对体外遗传毒理测试 Advantages and disadvantages 优点和缺点优点和缺点 Use of transgenic animal mutation assays 转基因动物致突变模型的应用转基因动物致突变模型的应用 Use for re

7、gulatory purposes 应用于规章的制定和法规的执行 Fill a gap in regulatory testing strategies 添补检测系统中的不足之处 Promotion by regulatory agencies 执法机构的促进推广 Tamoxifen Tumorigenesis in Experimental Animals 实验动物的三笨氧氨致癌性实验动物的三笨氧氨致癌性 =Rats Liver tumors and endometrial tumors =Mice Endometrial tumors O N OH O N OH O N N -Desmet

8、hyltamoxifen O NH Tamoxifen 4-Hydroxytamoxifen -Hydroxytamoxifen O N OH OAc O N Hydroxytamoxifen -Acetoxytamoxifen -(Deoxyguanosin- N2-yl)- dR O N NH NO NH NN DNA/hydrolysis Acetic anhydride/pyridine tamoxifen 0 50 100 600 900 1200 ConTam -OH Tam ENUConTam -OH Tam Mutants/106 plaques lacIcII Big Blu

9、e Rat - Liver Mutant Frequency Big Blue 大鼠肝大鼠肝lacI和和cII基因突变频率基因突变频率 a a,b a a a,b Mutation types 突变类型突变类型 Mutation Treatment Control Tamoxifen -Hydroxy tamoxifen C T 41 5 17 A G 3 5 0 G T 18 43 61 G C 11 3 10 A T 7 0 2 A C 3 5 0 Frameshift 13 25 10 Complex 4 14 0 Doses of AAs (mg/kg) 0246810 DNA add

10、ucts per 108 nucleotides 0 1000 2000 3000 4000 5000 6000 Aristolochic acids（AAs）-induced total DNA adducts 马兜铃酸的马兜铃酸的DNA加合物加合物 Kidney Liver AAs-induced cII mutant frequency (MFs) 马兜铃酸诱导的突变频率马兜铃酸诱导的突变频率 Doses of AAs (mg/kg) 0246810 Mutation frequency (X10-6) 0 500 1000 1500 Kidney Liver Autoradiograp

11、hic profiles of DNA adducts using 32P-postlabeling assay AB CD2 3 1 1 2 3A B CD2 3 1 1 2 3 spot 1: dA-AAI spot 2: dG-AAI spot 3: dA-AAII Kidney Liver Control AA (10mg/kg) Summary of the independent mutations in the liver and kiney cII gene from the AAs-treated and control Big Blue rats P = 0.106 P = 0