SELECTION MARKER GENES Positive selection: neomycin phosphotransferase(neo) hygromycin phosphotransferase(hyg) puromycin(pur) hypoxanthine phosphoribosyltransferase(hprt) Negative selection thymidine kinase(tk) cytosine deaminase(cd) hypoxanthine phosphoribosyltransferase(hprt
Positive selection: - neomycin phosphotransferase (neo) - hygromycin phosphotransferase (hyg) - puromycine (pur) - hypoxanthine phosphoribosyltransferase (hprt) Negative selection - thymidine kinase (tk) - cytosine deaminase (cd) - hypoxanthine phosphoribosyltransferase (hprt) SELECTION MARKER GENES
Positive selection Es cells that have the targeting vector: neo resistant Negative selection: Select against es cells with random vector integration sensitive to anti-herpes drugs, FIAU, ganciclovir 1mneo3}HS∨tk random 1HneoH3H+ random Molecular screen Eliminates random integrants without hsvtk Verifies recombination on both ends 3 probe 5 probe 1H neoh3 targeted allele 12}3} endogenous allele
Positive selection: ES cells that have the targeting vector: neo resistant Negative selection: Select against ES cells with random vector integration sensitive to anti-herpes drugs, FIAU, ganciclovir Molecular screen: Eliminates random integrants without HSVtk Verifies recombination on both ends 1 neo 3 HSVtk 1 neo 3 5’probe 1 2 3 targeted allele endogenous allele random 3’probe 1 neo 3 H random
Positive-Negative Selection RR112图[HR2 Genomic Locus STOP CODONS HR2 HHSY targeting HR1 Bae Vector H HR1HlB-geoH3HHR2 Mutant Locus
HR1 1 2 3 HR2 HR1 b-geo 3 HR2 HR1 b-geo 3 HR2 Genomic Locus Targeting Vector Mutant Locus HSVtk STOP CODONS Positive-Negative Selection
Screening ES Cell clones Screening Plate-Grow for DNA, restrction digest, then Southern o000000o Pck clones 000000000 and trypsinize 000000000 000000000 000000000 0o00000Oo 00000000o 000000000→000000000 0000000o0 000o0000 00000000O 000000000 00000000O 0000000o0 0000000 00000000o Master Plate. Freeze cells or continue to grow and
Screening Plate - Grow for DNA, restriction digest, then Southern Master Plate - Freeze cells or continue to grow and subplate Pick clones and trypsinize plate and grow Screening ES Cell Clones
Strategies for Screening HR1H1H2H:3 HR2 Genomic Locus A ⊥[HR1HB-9oLHR2 HSV targeting H HR1 H:- HR2 Mutant Locus SOUTHERN BLOT SCREENING: A-A, C-C= both WT and MT, but different PCR SCREENING: sizes, when probed with 5' and 3 probes, respectiv 1-2= WT locus A-B, B-C Specific to MT when probed with the same probes
HR1 1 2 3 HR2 HR1 b-geo 3 HR2 HR1 b-geo 3 HR2 Genomic Locus Targeting Vector Mutant Locus HSVtk 1 2 3 PCR SCREENING: 1-2 = WT locus 1-3 = MT locus A A A A A A C C C C C B 5’ 3’ SOUTHERN BLOT SCREENING: A-A, C-C = both WT and MT, but different sizes, when probed with 5’ and 3’ probes, respectively A-B, B-C = Specific to MT when probed with the same probes Strategies for Screening