In the JHU Transgenic Core Laboratory, 50 ng/ul φC31mRNA and 3 ng/ul TRE-hShh targeting vector were diluted in RNAse free injection buffer (10 mM Tris–HCl, pH 7.4, 0.25 mM EDTA) and injected into the pronucleus of 297 zygotes from Rosa26 TARGATT mice (Applied StemCell). The embryos were transferred into the oviducts of 11 pseudopregnant ICR moms. Founder pups were identified by PCR of tail DNA using primer sets SSL and SSR (Applied StemCell) that were specific for the right and left junctions of the attP/Rosa26 locus. Three mice positive for the insertion were identified among 31 pups. Two lines had the expected attP1/attP3 insertions, while the third contained only the attP3 insertion.
We verified founders using Southern blot analysis. The 5’- and 3’- probes were made by PCR with primer sets Tsh5prF/Tsh5prR of WT genomic DNA and Tsh3prF2/Tsh3prR3 of pROSA26-PA (plasmid #21,271, Addgene), respectively, and then labeled with α-32P-dCTP (Amersham Rediprime II Random Prime Labelling System). Genomic DNA from the 3 founder lines and controls were digested with HindIII, separated on 1 × TBE 0.9% agarose gel at 50 V overnight with recirculation, transferred to Hybond N + , and hybridized with 5ʹ- or 3ʹ- probes. The membrane was imaged on Molecular Dynamics Storm Imager and then exposed to film.
We verified founders using Southern blot analysis. The 5’- and 3’- probes were made by PCR with primer sets Tsh5prF/Tsh5prR of WT genomic DNA and Tsh3prF2/Tsh3prR3 of pROSA26-PA (plasmid #21,271, Addgene), respectively, and then labeled with α-32P-dCTP (Amersham Rediprime II Random Prime Labelling System). Genomic DNA from the 3 founder lines and controls were digested with HindIII, separated on 1 × TBE 0.9% agarose gel at 50 V overnight with recirculation, transferred to Hybond N + , and hybridized with 5ʹ- or 3ʹ- probes. The membrane was imaged on Molecular Dynamics Storm Imager and then exposed to film.