Generation of transgenic mice and mouse genetics
Er81EWS-Pea3 mice were generated following a strategy similar to that described for the generation of Er81NLZ mice [14]. In brief, a targeting vector with a cDNA coding for EWS-Pea3 was inserted in frame with the endogenous start ATG into exon 2 of the Er81 genomic locus and used for homologous recombination in W95 ES cells. EWS-Pea3 represents a fusion gene between the amino terminal of EWS and the ETS domain of Pea3 [20]. The primer pair used to specifically detect the Er81EWS-Pea3 allele was 5′- CAGCCACTGCACCTACAAGAC-3′ and 5′- CTTCCTGCTTGATGTCTCCTTC-3′. For the generation of TaumGFP and TauEWS-Pea3 mice, lox-STOP-lox-mGFP-IRES-NLS-LacZ-pA and lox-STOP-lox-EWS-Pea3-IRES-NLS-LacZ-pA targeting cassettes were integrated into exon 2 of the Tau genomic locus (the endogenous start ATG was removed in the targeting vectors; details available upon request). mGFP was provided by P. Caroni [25]. ES cell recombinants were screened by Southern blot analysis using the probe in the 5′ region as described previously [41]. Frequency of recombination in 129/Ola ES cells was approximately 1/3 for both Tau constructs. For the generation of PVCre mice, mouse genomic clones were obtained by screening a 129SV/J genomic library (Incyte, Wilmington, Delaware, United States). For details on the genomic structure of the mouse PV locus see [42]. An IRES-Cre-pA targeting cassette [33] was integrated into the 3′ UTR of exon 5, and ES cell recombinants were screened with a 5′ probe (oligos, 5′- GAGATGACCCAGCCAGGATGCCTC-3′ and 5′- CTGACCACTCTCGCTCCGGTGTCC-3′; genomic DNA, HindIII digest). The frequency of recombination in 129/Ola ES cells was approximately 1/20. Recombinant clones were aggregated with morula stage embryos to generate chimeric founder mice that transmitted the mutant alleles. In all experiments performed in this study, animals were of mixed genetic background (129/Ola and C57Bl6). Thy1spGFP transgenic mice were generated in analogy to De Paola et al. [25], and for these experiments a strain of mice with early embryonic expression was selected. Isl1Cre and Hb9Cre mouse strains have been described [33,43] and Bax+/− animals were from Jackson Laboratory (Bar Harbor, Maine, United States) [27]. Timed pregnancies were set up to generate embryos of different developmental stages with all genotypes described throughout the study.