Gene targeting in embryonic stem cells Targeting vector with the desired change is electroporated into Es cells Homologous recombinants are identified by drug selection &molecular screening Recombinant es clones are injected into blastocysts to produce chimeric mice Chimeras are bred to produce heterozygotes Heterozygotes are intercrossed to produce homozygous mutants
Gene targeting in embryonic stem cells • Targeting vector with the desired change is electroporated into ES cells • Homologous recombinants are identified by drug selection &molecular screening • Recombinant ES clones are injected into blastocysts to produce chimeric mice • Chimeras are bred to produce heterozygotes • Heterozygotes are intercrossed to produce homozygous mutants
打靶载体的设计 选用同源DNA 同源臂的长度 正、负选择
打靶载体的设计 • 选用同源DNA • 同源臂的长度 • 正、负选择
Factors influencing targeting efficiency isogenic DNA (perfect homology) 10-25 fold van Deursen J, Wieringa B Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors. Nucleic Acids Res. 20: 3815-20, 1992 size of region of homology exponential relationship Capecchi MR. Altering the genome by homologous recombination Science.244:1288-9,1989 robust screen Positive controls
Factors influencing targeting efficiency • isogenic DNA (perfect homology) 10-25 fold van Deursen J, Wieringa B. Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors. Nucleic Acids Res. 20:3815-20, 1992. • size of region of homology exponential relationship Capecchi MR. Altering the genome by homologous recombination. Science. 244:1288-9, 1989 • robust screen! Positive controls
Homologous recombination 1-Length of homologous sequences 2-Isogenic DNA Hom. Rec Efficiency Base pair 25bp 2000bp
Homologous recombination 1- Length of homologous sequences Hom. Rec. Efficiency Base pair 25bp 2000bp 2- Isogenic DNA
rene targeting H NEOMYCIN HHH H NEOMYCIN HHHH Total 4 Kbp (each arm not less than 500bp) Delete coding sequences Change reading frame Transcription of neo in antisense direction
NEOMYCIN X X Gene targeting NEOMYCIN Total 4 Kbp (each arm not less than 500bp) Delete coding sequences Change reading frame Transcription of Neo in antisense direction