ES cell electroporation and production of chimeras was performed by the University of Connecticut Gene Targeting and Transgenic Facility (GTTF). The pR26-CLNFZG targeting vector was linearized with SfiI and electroporated into 129S6/C57BL/6 hybrid ES cells (D1: established by GTTF). Southern blot hybridization on BamHI-digested genomic DNA was used to screen for homologous recombination on the 5′ end using a 0.67 kb probe outside of the 5′ homology arm. The probe was liberated by EcoRV digestion of the PCR product generated with the following primers: 5′-TTCCTCTCAATATGCTGCACACAAA-3′ and 5′-GCCCAGAGAGAAAGGCTCTCCTTCA -3′. The targeted and wild-type alleles produced products of 11.5 kb and 5.8 kb, respectively. Nested PCR was used to assay for correct targeting on the 3′ end. The targeted allele generated a 5.8 kb diagnostic fragment with the following primers; 1st PCR, 5′-GGGAAGACAATAGCAGGCATGCTGG-3′ and 5′-GATGCCCAATTCCAACTGTGAAGAC-3′; 2nd PCR, 5′-TTCTGAGGCGGAAAGAACCAGCTAG-3′ and 5′-TTCCTCTCAATATGCTGCACACAAA-3′.
Chimeric mice were produced from two targeted ES cell clones by aggregation with CD1 embryos. Germ line transmission of the targeted allele was assessed by LacZ PCR with primers (5′-GCGGATCCGAATTCGAAGTTCC-3′ and 5′-TGGGTCTCCAAAGCGACTCC-3′) that generate a 333-bp product. R26ZG was generated by a cross between R26NZG/+ and Hprt1Cre/+ mice. Removal of the PGKNEO cassette was verified by the presence of a 193-bp PCR product using primers that flank PGKNEO (5′-ACTGGGCACAACAGACAATCG-3′ and 5′-GCTTCAGTGACAACGTCGAG-3′). R26NG was generated by a cross between R26NZG/+ and R26FLPe/FLPe mice. As R26FLPe-driven excision of the nlslacZ cassette was incomplete, the resulting mosaic F1 offspring were crossed with R26FLPe/+ mice to establish the R26NG line. Removal of the nlslacZ cassette was assessed by PCR with a forward PGK pA cassette primer (5′-GATCAGCAGCCTCTGTTCCACA-3′) and a reverse EGFP primer (5′-CGCTGAACTTGTGGCCGTTTAC-3′) that amplifies a 264-bp product. Lines were maintained by breeding to FVB mice.
Chimeric mice were produced from two targeted ES cell clones by aggregation with CD1 embryos. Germ line transmission of the targeted allele was assessed by LacZ PCR with primers (5′-GCGGATCCGAATTCGAAGTTCC-3′ and 5′-TGGGTCTCCAAAGCGACTCC-3′) that generate a 333-bp product. R26ZG was generated by a cross between R26NZG/+ and Hprt1Cre/+ mice. Removal of the PGKNEO cassette was verified by the presence of a 193-bp PCR product using primers that flank PGKNEO (5′-ACTGGGCACAACAGACAATCG-3′ and 5′-GCTTCAGTGACAACGTCGAG-3′). R26NG was generated by a cross between R26NZG/+ and R26FLPe/FLPe mice. As R26FLPe-driven excision of the nlslacZ cassette was incomplete, the resulting mosaic F1 offspring were crossed with R26FLPe/+ mice to establish the R26NG line. Removal of the nlslacZ cassette was assessed by PCR with a forward PGK pA cassette primer (5′-GATCAGCAGCCTCTGTTCCACA-3′) and a reverse EGFP primer (5′-CGCTGAACTTGTGGCCGTTTAC-3′) that amplifies a 264-bp product. Lines were maintained by breeding to FVB mice.
