Conditional knockout and genotyping strategies.

(A) Schematic version of the strategy used for the generation of the floxed Rfx4 KO mice. In the WT allele, exon 4 was chosen for deletion. In the floxed allele, two loxP sites flanked exon 4, and a neomycin (neo) cassette with two Frt sites was inserted. Cre recombinase causes deletion of exon 4. “G1F”, on the 5’ arm, and “G1R”, on the 3’ arm, are genotyping PCR primers which produce WT, flox or KO bands. (B) Genotyping PCR: Lane +/- refers to a Rfx4^+/- mouse, and the two bands were from floxed and KO alleles from a KO mouse; lane f/+ refers to a control mouse, and the two bands were from floxed and WT alleles from a Rfx4^f/+mouse. (C) RT-PCR demonstrated a lower band in Rfx4^+/- brain and spinal cord, representing the transcript produced by the KO allele. (D) As determined by qPCR, Rfx4 mRNA levels were decreased by approximately 50% (P< 0.01, n = 4) in the brains of Rfx4^+/- mice, as determined by fold changes after normalizing with Gapdh mRNA. (E) Image of the domed head of a mouse at 4 weeks of age (left) that was heterozygous for the Rfx4 KO.