转基因治病模型

Click to edit Master title style,Click to edit Master text styles,Second Level,Third Level,Fourth Level,Fifth Level,Transgenic Mutation Animal Assays,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,Introduction,Transgenic mutation systems,Development of transgenic mutation animals,Protocol for transgenic mutation assays,Validation of transgenic mutation assays,Comparison of mutations in transgenes and native genes,Transgenic mutation assays vs.other genotoxic assays,and vs.carcinogenicity assays,Use of transgenic animal mutation assays,Regulatory use for,in vivo,genotoxicity testing,Mechanistic,studies,Conclusion,Introduction,导言,What is Mutation?,什么是突变？,Mutations are heritable changes in the nucleotide sequence of DNA,Mutations in somatic cells can cause neoplasms and possibly aging,Mutations in germ cells are responsible for a large number of genetic diseases,including birth defects and cancer,Mutation and Cancer,突变和癌症,Mutation is not equal to cancer,Cancer:a disease caused by somatic cell mutations,Mutation as a biomarker of cancer,突变可用作癌症的生物指标,Mutation assays as short-term tests for carcinogenicity,In vitro:,Salmonella,mutation assays and gene mutations in cultured mammalian cells.,In vivo:,Hprt,assay,Aprt,assay,Tk,assay and transgenic assay,In vivo evidence is more important than in vitro evidence,Transgenic mutation systems,转基因致突变系统,Transgenic animals for mutation assays,检测突变的,转基因动物,Transgenic animals are animals whose cells contain foreign target genes(reporter genes),reporter genes are normally located on shuttle vectors that are derivatives of bacteriophage or plasmids,The vectors recover the mutational target from rodents for mutant detection in bacteria,Shuttle vector,穿梭,载体,A shuttle vector containing reporter gene(s)is constructed using recombinant DNA technologies.Following is a,LIZ shuttle vector used for Big Blue transgenic rodents,c,lacZ,lacI,Amp,r,S,L,cos,cI,857,cro,P,RE,O,P,Q,A J,cos,cII,Q,R,P,R,Transgenic rodent mutation models,转基因动物,突变模型,Big Blue Rats,大蓝大鼠,Isolation of genomic DNA,Treatment,Mutant manifestation,Collection 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,C,olor selection,lacZ,lacI,Procedure for transgenic rodent mutation assays,转基因动物致,突变实验步骤,Treatment,药剂处理,For regulatory safety assessment,the International Workshop on Genotoxicity Test Procedures,(IWGT)recommends repeat treatment of animals for 28 days with the maximum tolerated dose(MTD),Two further dose groups receiving 1/3 and 2/3 the MTD are desirable in case that the number of animals in the high dose group is reduced by agent toxicity,If there are no significant differences between the sexes in terms of toxicity or metabolism,the IWGT recommends that male animals should normally be used,Mutant manifestation time,突变体显示时间,Mutant manifestation time is the period between the last treatment and the time of sampling tissues for mutation,The manifestation time is dependent on the tissue that is assayed,the IWGT recommends a general schedule of sampling 3 days after 28 days of repeated treatment for all tissues,Collection of tissues,组织收集,All potential target tissues from which reasonable amounts of DNA can be recovered should be collected,To minimize the in situ degradation of the DNA,each tissue should be dissected,wrapped,and flash-frozen in liquid nitrogen,Store the frozen tissues at 80C,Isolation of genomic DNA,分离染色体组,DNA,Genomic DNA should be 100300 kb or larger for efficient phage rescue,The RecoverEase DNA isolation kit(Stratagene)is recommended for the isolation of high molecular genomic DNA,Genomic DNA can be stored in a TE buffer at 4,0,C,protected from light,for up to one month,Statistics and criteria,统计和准则,200,000,transgenes per animal from at least 5 animals per treatment must be screened to detect a 2-fold increase in the MF,If an appropriate statistical analysis is performed,the difference between the MFs from the treated and control groups can be used to judge a testing agent positive or negative,Validation of Transgenic Systems,转基因系统的,确证,Do the test systems measure what we are intending to measure?,Are the assays predictive of the endpoints of interest?,MFs in Lymphocyte,Hprt,and,lac,I vs.Liver,lac,I 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变频率,MF(x 10,-6,),Hprt,vs.,lac,I 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,Mutagen,ENU,DMBA,BaP,Thiotepa,EO,None,Model,Mouse,Rat,Mouse,Rat,Mouse,Rat,Gene,Hprt,lacI,Hprt,lacI,Hprt,lacI,Hprt,lacI,Hprt,lacI,Hprt,lacI,Induced mutant frequency(x 10,-6,),56,80,160,316,9,138,39,106,12,0,0,0,%,mutation at G:C sites,9,41,38,41,ND,92,75,88,36,72,75,80,%,at CpG sites,0,13,3,6,ND,79,17,65,ND,44,22,70,%,frameshifts/small deletions,18,4,3,13