转基因治病模型学习教案

1、会计学1第一页，共40页。第1页/共40页第二页，共40页。第2页/共40页第三页，共40页。nMutations are heritable changes in the nucleotide sequence of DNAnMutations in somatic cells can cause neoplasms and possibly agingnMutations in germ cells are responsible for a large number of genetic diseases, including birth defects and cancer第3页/共4

2、0页第四页，共40页。第4页/共40页第五页，共40页。第5页/共40页第六页，共40页。第6页/共40页第七页，共40页。第7页/共40页第八页，共40页。clacZlacIAmprSLcoscI857croPREOPQA JcoscIIQRPR第8页/共40页第九页，共40页。第9页/共40页第十页，共40页。第10页/共40页第十一页，共40页。Isolation of genomic DNATreatmentMutant manifestationCollection of tissuesPhage packaging or plasmid recovery Infection of

3、E. coli Plating and plaque formationTiter plate Mutant platePositive selectionSequencing of mutantsCalculation of mutant frequencyColor selection lacZ lacIProcedure for transgenic rodent mutation assays 转基因动物致突变实验转基因动物致突变实验(shyn)步骤步骤第11页/共40页第十二页，共40页。第12页/共40页第十三页，共40页。第13页/共40页第十四页，共40页。第14页/共40页第

4、十五页，共40页。第15页/共40页第十六页，共40页。第16页/共40页第十七页，共40页。第17页/共40页第十八页，共40页。MF (x 10-6)第18页/共40页第十九页，共40页。MF (x 10-6)第19页/共40页第二十页，共40页。Comparisons of mutations in the Hprt and lacI genesHprt和lacI基因突变(j yn t bin)的比较MutagenENU DMBA BaP Thiotepa EO None ModelMouseRatMouseRatMouseRatGeneHprtlacIHprtlacIHprtlacIH

5、prtlacIHprtlacIHprtlacIInduced mutant frequency (x 10-6)568016031691383910612000% mutation at G:C sites9413841ND92758836727580% at CpG sites01336ND791765ND442270% frameshifts /small deletions184313ND728 3439141710Strand bias of mutationsyesnoyesnoyesnoyesnoNDNDnono第20页/共40页第二十一页，共40页。Transgenic mous

6、e mutation assay vs. mouse bone marrow micronucleus test for predicting mouse carcinogenicity 小鼠致癌性的预测：转基因小鼠致突变测试小鼠致癌性的预测：转基因小鼠致突变测试(csh)对小鼠骨髓微核体测试对小鼠骨髓微核体测试(csh)Term#Calculation for the mouse bone marrow micronucleus testCalculation for MutaTMmouse and/or Big Blue mouseSensitivity63% (24/38)82% (31

7、/38)Specificity0% (0/2)100% (2/2)Positive predictivity92% (24/26)100% (31/31) Negative predictivity0% (0/14)22% (2/9)Overall accuracy60% (24/40)83% (33/40)第21页/共40页第二十二页，共40页。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致突变结果比较：转基因动物致突变测试(csh)对体外遗传毒

8、理测试对体外遗传毒理测试(csh)TG assaysResults on genotoxicity in vitroResultsNumber of sub-stancesGene mutation Chromsomal aberrationDirect or indirect measure for DNA damage+-Inconc.nd/na+-Inconc.nd/na+-Inconc.nd/naTG+42 3633028 24832433TG-13652053415431第22页/共40页第二十三页，共40页。第23页/共40页第二十四页，共40页。第24页/共40页第二十五页，共4

9、0页。第25页/共40页第二十六页，共40页。第26页/共40页第二十七页，共40页。第27页/共40页第二十八页，共40页。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=RatsLiver tumors and endometrial tumors =MiceEndometrial tumors第28页/共40页第二十九页，共40页。ONOHONOHONN -DesmethyltamoxifenONHTamoxifen4-Hydroxytamoxifen -Hydroxytamoxifen第29

10、页/共40页第三十页，共40页。ONOHOAcON Hydroxytamoxifen -Acetoxytamoxifen -(Deoxyguanosin- N2-yl)-dRONNHNONHNNDNA/hydrolysisAcetic anhydride/pyridinetamoxifen第30页/共40页第三十一页，共40页。0501006009001200ConTam -OHTamENUConTam -OHTamMutants/106 plaqueslacIcIIBig Blue Rat - Liver Mutant FrequencyBig Blue 大鼠肝大鼠肝lacI和和cII基因突

11、变频率基因突变频率aa,baaa,b第31页/共40页第三十二页，共40页。Mutation types 突变突变(tbin)类型类型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 第32页/共40页第三十三页，共40页。Doses of AAs (mg/kg)0246810DNA adducts per 108 nucleoti

12、des0100020003000400050006000Aristolochic acids（AAs）-induced total DNA adducts马兜铃酸的马兜铃酸的DNA加合物加合物KidneyLiver第33页/共40页第三十四页，共40页。AAs-induced cII mutant frequency (MFs)马马兜兜铃铃酸酸诱导诱导(yudo)的突的突变频变频率率Doses of AAs (mg/kg)0246810Mutation frequency (X10-6)050010001500KidneyLiver第34页/共40页第三十五页，共40页。Autoradiographic profiles of DNA adducts using 32P-postlabeling assay spot 1: dA-AAI spot 2: dG-AAI spot 3: dA-AAIIKidneyLiverControl AA (10mg/kg)第35页/共40页第三十六页，共40页。Summary of the independent mutations in the liver and kiney cII gene from the AAs-treated and control Big Blue ratsP = 0.106 P = 0.065 第36页/