Figure 2.
Prx1 limb enhancer replacement and deletion by gene targeting. (A) Strategy for generating targeted alleles. The genomic region surrounding the Prx1 limb enhancer is diagrammed schematically. The targeting vector possesses 7.05 kb of total homology in three parts: 3.8 kb 5′, 2.2 kb central, and 1.05 kb 3′ homology (indicated by the thick line in wild-type allele). A PGKneobpA-lox neo expression cassette (neo) was introduced into the BamHI site of intron 1 separating the central and 3′ homologies. The neo cassette introduces a StuI restriction site used for Southern analysis. A MCTK1 expression cassette (tk) was included for negative selection. The Carollia limb enhancer (Cp) was introduced on a 1-kb HindIII restriction fragment replacing the equivalent 1-kb segment surrounding the endogenous enhancer (Mm) and separating the 5′ and central homologies (BatE targeting vector). An identical vector was also constructed but with the 1-kb enhancer segment deleted and replaced with a HindIII restriction site for Southern analysis (EnhΔ targeting vector). The positions of the external 5′ and 3′ probes used for Southern analysis to screen for targeted ES cell clones are shown below the wild-type allele. The sizes of the expected restriction fragments recognized by the probes are indicated above each allele. The neo cassettes were removed in vivo by Cre expressed from an ectopic male germline-specific Prm1-Cre transgene. (B) Southern analysis of bat enhancer replacement targeted ES cell clones. Targeted clones digested with HindIII produce 23-kb wild-type and 5.7-kb targeted bands when hybridized with an external 5′ probe. Targeted clones digested with StuI produce 7.6-kb wild-type and 3.9-kb targeted bands when hybridized with an external 3′ probe. (C) Southern analysis of Prx1BatE mice: Genomic DNA extracted from Prx1BatE/+ mice digested with HindIII and hybridized with an external 5′ probe produces 23-kb wild-type and 5.7-kb targeted bands; Prx1BatE/BatE mice produce only the 5.7-kb targeted band. Genomic DNA extracted from Prx1BatE/+ mice digested with BamHI and hybridized with an external 3′ probe produces 5.6-kb wild-type and 21.5-kb targeted bands when hybridized with an external 3′ probe; Prx1BatE/BatE mice produce only the 21.5-kb targeted band. (D) Southern analysis of enhancer deletion targeted ES clones. Targeted clones digested with HindIII produce 23-kb wild-type and 5.7-kb targeted bands when hybridized with an external 5′ probe. Targeted clones digested with StuI produce 7.6-kb wild-type and 3.9-kb targeted bands when hybridized with an external 3′ probe. (B) BamHI; (H) HindIII; (N) NsiI; (S) SalI; (St) StuI.