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Supplementary Fig.1_Haigh. RE cloning. PCR. BP-Reaction. A. L1. L2. cDNA. 0. pEntry Clone. Km R. LR reaction Bacterial DNA analysis and expansion Sequence validation and linearization. 28°C. 1.3 kb. 7-10 days. G1. SA. S1. B1. B2. Target vector. Pvu I. 5’. IRES-eGFP.
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Supplementary Fig.1_Haigh RE cloning PCR BP-Reaction A L1 L2 cDNA 0 pEntry Clone KmR LR reaction Bacterial DNA analysis and expansion Sequence validation and linearization 28°C 1.3 kb 7-10 days G1 SA S1 B1 B2 Target vector PvuI 5’ IRES-eGFP PGK-DTA PGK-neo-STOP 3’-ROSA26 homology arm cDNA PvuI 10 loxP loxP S2 B G2 7-10 days ES cell electroporation Drug Selection Neo resistant G4 ES cell clones 20 ES Colony Picking C PCR Screening analysis 7-10 days Freeze 2X Master Plates + + + 1.3 kb PCR Screen 96 Well Format ES cell clone expansion and analysis Expand PCR positive clones DNA isolation for Southern 5’, 3’ and single copy integration 1 Time in Days 30 D Southern Confirmed ROSA26 conditional ES Cell clones LN2 Cre excision Puro-Selection Expand and Freeze Confirmed clones 7-10 days F Diploid Aggregation 2 LN2 Southern Confirmed ROSA26 Expressing ES Cell clones G 90 X 60-70 days Q-RT-PCR & Western analysis E 100% Germline Transmitting G0 ROSA26 Conditional Male Tetraploid Aggregation Tissue-specific Cre X Embryo ubiquitously expressing ROSA26 Tg Embryos expressing ROSA26 Tg in cell/tissue specific manner 100 Sox-2 Cre (Ubiquitous Expression)
