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Fig. 2. Conditional inactivation of the GFR1 gene in mouse. (A) Schematic drawing of the GFR1 locus, the floxed GFR1 and GFP knock-in (null) alleles. Floxed GFR1 allele expresses GFR1 cDNA, thereby serving as a functional allele. Activation of Cre recombinase results in a removal of floxed GFR1, simultaneously generating GFP knock-in (GFR1-null) allele. (B) Schematic diagram of breeding strategy. GFR1^+/-:CAGGCre-ER^TM mice were mated to GFR1^flox/flox mice to obtain GFR1^flox/+:CAGGCre-ER^TM (control) and GFR1^flox/-:CAGGCre-ER^TM (knockout, cKO) embryos in the same litter. Pregnant mice were given an intraperitoneal injection of 4-OHT at the desired time point to induce Cre activity. Following recombination, GFP reporter gene was expressed in the control and cKO embryos. (C) 4-OHT-induced Cre recombination in enteric neurons. Minimal `leaky' recombination in the colon of E15.5 GFR1^flox/+ embryo was observed in the absence of 4-OHT treatment (left panel). Administration of 4-OHT induced GFP expression in a large number of neurons in the colon, indicating that Cre recombination occurred in the majority of enteric neurons in the colon (right panel). (D) Histological analysis of cKO enteric plexus showing no GFR1-immunoreactivity (red) in GFP⁺ cells (green). Note the expression of GFR1 (red) in neighboring un-recombined cells. (E) Absence of pERK in GFR1-deficient enteric neurons. Activation of ERK (pERK) was detected in GFP-negative-TuJ1-positive cells (arrowhead). By contrast, no pERK signal was observed in GFP-positive-TuJ1-positive cells (asterisk). Scale bars: 20 µm in C; 5 µm in D,E.

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