Figure 1.
Targeting the Cald1 gene.(A) Using clone RPCI23–104H13 as Cald1 gene template, the Cald1 BAC targeting construct was generated by bacterial engineering, linearized with NotI, and electroporated into V6.5 ES cells. (B) DNA of G418 selected ES cell clones were isolated, digested by the endonuclease BamHI, and analyzed by semiquantitative Southern blot using a probe against Col12a1 for signal standardization. The internal Cald1 probe showed signal intensity reduced by half in ES cell clones with homologous recombination events. (C) Genotype of the offspring from mating Cald1+/− × Cald1+/− was determined in two separate PCR reactions. The primers for the WT band were located within the ablated Cald1 sequence (lanes 1, 3, and 5); one of the primers for detection of the homologous recombination event was located within the neomycin selection cassette, and the other in the Cald1 gene (lanes 2, 4, and 6). (D and E) Representative Western blots from lysates from E18.5 ileal smooth muscle (D), urinary bladder, kidney, liver, and mesentery (E) using antibodies against CaD and GAPDH (loading control) and smooth muscle α-actin (α-smAct). In liver, kidney, and mesentery artery, CaD is less abundant than in bladder. To get a signal intensity from these tissues comparable to that of bladder, 40 times more protein had to be loaded. No CaD signal was detected in KO (Cald1−/−) tissue; signal intensity was reduced in HET ileum (Cald1+/−) compared with WT littermates (Cald1+/+). Expression of GAPDH was not affected by the mutation.

Targeting the Cald1 gene. (A) Using clone RPCI23–104H13 as Cald1 gene template, the Cald1 BAC targeting construct was generated by bacterial engineering, linearized with NotI, and electroporated into V6.5 ES cells. (B) DNA of G418 selected ES cell clones were isolated, digested by the endonuclease BamHI, and analyzed by semiquantitative Southern blot using a probe against Col12a1 for signal standardization. The internal Cald1 probe showed signal intensity reduced by half in ES cell clones with homologous recombination events. (C) Genotype of the offspring from mating Cald1+/− × Cald1+/− was determined in two separate PCR reactions. The primers for the WT band were located within the ablated Cald1 sequence (lanes 1, 3, and 5); one of the primers for detection of the homologous recombination event was located within the neomycin selection cassette, and the other in the Cald1 gene (lanes 2, 4, and 6). (D and E) Representative Western blots from lysates from E18.5 ileal smooth muscle (D), urinary bladder, kidney, liver, and mesentery (E) using antibodies against CaD and GAPDH (loading control) and smooth muscle α-actin (α-smAct). In liver, kidney, and mesentery artery, CaD is less abundant than in bladder. To get a signal intensity from these tissues comparable to that of bladder, 40 times more protein had to be loaded. No CaD signal was detected in KO (Cald1−/−) tissue; signal intensity was reduced in HET ileum (Cald1+/−) compared with WT littermates (Cald1+/+). Expression of GAPDH was not affected by the mutation.

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