化学遗传学筛选应用：斑马鱼突变体突变基因的定位 Positional cloning (mapping) of mutant,张文清 教授 医学院/发育生物学团队,Large-scale Forward Genetic Screening （2008-） NR/SBB/e1/rag1,Mesoderm （腹侧中胚层）,Hemogenic Endothelium （血源性血管内皮细胞）,HSC （造血干细胞）,Lymphoid progenitor（淋系祖细胞）,Erythrocyte（红细胞）,Macrophage（巨噬细胞）,Lymphocyte（淋巴细胞）,Granulocyte（粒细胞）,GMP （单核巨噬祖细胞）,pu.1,I: 多谱系发育缺陷 （17个突变体） III: 巨噬-中性粒细胞缺陷 （1个突变体） IV: 巨噬细胞缺陷 （5个突变体） V: 中性粒细胞缺陷 （2个突变体）,cloche、scl、cmyb,I,II,III,IV,V,coronin1a,sae1,JGG Plos One Blood Leukemia Development Development Blood JBC,36mutants cloche cmyb pu.1 sae1 scl runx1/pu.1 coronin1a,2012 2011 2012 2013 2012 2013 2013 2012,Analysis of the Development of Hematopoiesis（2011-）,350,CMP （髓系祖细胞）,350,Sudan black B staining,sibling,350 mutant: Sudan black B staining is negative,3dpf,3dpf,Neutral red staining,4dpf,4dpf,sibling,350,350 mutant: The expressions of mpx and lyz are not affected , but MPO is enzymatically inactive,lyz,2dpf,2dpf,350 mutant: Few granules in the neutrophils are detected,350,350 mutant: Primitive hematopoiesis is not affected,sibling,350,22hpf,22hpf,22hpf,22hpf,22hpf,22hpf,36hpf,2dpf,2dpf,c-myb,c-myb,sibling,350,350 mutant: Definitive HSCs are not affected in the VDA,36hpf,Phenotype,Chromosome,Gene,Initial mapping,Fine mapping,Positional cloning (mapping) of 350,+,-,+,+,-,+,+,+,+,+,-,+,-,-,-,+,F 0,F 2,F 1,AB,WIK,Mutant pool,Sibling pool,Bulked segregant analysis (BSA) 群组分离分析法,Molecular markers for BSA: RFLPs(Restriction Fragment Length Polymorphisms) RAPDs(Random Amplified Polymorphism DNAs) SSLPs(Simple Sequence Length Polymorphisms),5-GTGGAGGTGTTGGGTTTGTACT(GTGTGTGTGTGTGTGTGTGTGTGTGTGT)ACTTGCGGATGATCTCTGCAGCATGATGTA -3,5-GTGGAGGTGTTGGGTTTGTACT(GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT)ACTTGCGGATGATCTCTGCAGCATGATGTA -3,(GT)100,(GT)110,homologous chromosome (AB background),homologous chromosome (wik background),AB band,WIK band,AB WIK Mutant Sibling,AB WIK Mutant Sibling,Linked marker,Unlinked marker,Marker 1 Marker 2,AB WIK,z10919 on chromosome 11 appeared to be linked with 350 mutational site,North,MGH panel, chromosome: 10, units: cM,MGH panel, chromosome: 11, units: cM,AB grandparents,AB grandparents,Wik grandparents,Wik grandparents,Mutant pool,Mutant pool,Sibling pool,Sibling pool,Genetic distance,Recombination rate,Recombinant,80,15,5,Recombination rate (R) =(15+52)/200=12.5%,Single mutant embryo,100,Marker 1 Marker 2 Marker 3,AB WIK,10,6,1,1,1,1,Marker 1,Marker 2,Marker 3,R1=3/20=15%,R2=2/20=10%,R3=1/20=5%,Single mutant embryo,Recombinant of marker 1,Recombinant of marker 2,Recombinant of marker 1,Recombinant of marker 2,North or south ?,z3362,z10919,z3362 and z10919 are on the same side of 350 mutational site,AB,wik,AB,wik,Single mutant embryo,Single mutant embryo,350 mutational site is close to the north end of chromosome 11,z10919,z1445,z1445 and z10919 are on the same side of 350 mutational site,Recombinants of z10919,Recombinants of z10919,AB,wik,AB,wik,350 mutational site is close to the north end of chromosome 11,z10919,FP326665-13,350 mutational site is located between z10919 and FP 326665-13,AB,wik,Single mutant embryo,Single mutant embryo,AB,wik,CU633745-3,Recombinants of z10919 (32/3120),CU929297-13,350 mutational site locates between CU633745 and CU929297,Recombinants of FP326665-13 (3/3120),Genetic interval showing seven gene candidates,Exon sequence:,Four bases-insertion were found in poc1a,Mutant,A highly conserved Poc1a in zebrafish,DrPoc1a,Fourrage, C., S. Chevalier and E. Houliston, A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies. PLoS One, 2010. 5(11): p. e13994.,SOFT (short stature, onychodysplasia, facial dysmorphism, and hypotrichosis),poc1a cas9,WT cut,WT pcr,