Generation of the Tbx1-GFP mouse. (A) Tbx1-GFP was inserted into the multiple cloning site (MCS) of pBigT downstream of a triple polyadenylation sequence (tpA) that is flanked by LoxP sites (triangles). Additional subcloning into the pROSA26PA plasmid resulted in the final targeting construct. 5’ and 3’ homology arms mediate recombination with the endogenous Rosa26 locus. (B) Whole cell lysates from COS7 cells transfected (T) with Tbx1-GFP plasmid or untransfected (UT). The fusion protein is ≈75 kD and is detected by anti-GFP and anti-Tbx1 on Western blot. (C) Southern blot was used to screen ESC clones. Genomic DNA was linearized with EcoRV and hybridized with a radiolabeled probe that binds upstream of the 5’ homology arm. Two of five positive clones are shown. The correctly targeted allele is 4,071 bp and the wildtype allele is 11,517 kb.
Freyer et al. BMC Developmental Biology 2013 13:33 doi:10.1186/1471-213X-13-33