Fig. 1.

Targeting the Rpl22 genomic locus. (A) Targeting strategy for the genomic locus of Rpl22. A loxP site was inserted 5′ to the wild-type exon 4. A loxP-FRT-neomycin resistance-FRT cassette was inserted 3′ to the wild-type exon 4 followed by a modified Rpl22 exon 4 containing the HA epitope tag inserted before the Rpl22 stop codon. F1 heterozygous offspring were bred to FLPeR mice to remove the neomycin cassette used for selection in the ES cells. Crossing the RiboTag mouse to a Cre recombinase-expressing mouse results in deletion of the wild-type exon 4 in the target cell population and replacement with the Rpl22^HA exon 4. (B) Southern blot strategy used to identify correctly targeted ES cells. The wild-type HindIII/SfiI DNA fragment is 9,908 bp, while the correctly targeted HindIII/SfiI fragment is 8,749 bp. (C) PCR products using oligonucleotides that amplify the loxP-containing intron sequence 5′ to the wild-type exon 4 of the Rpl22 gene. The wild-type PCR product is 260 bp, while the mutant PCR product is 290 bp. (D) Western analysis of wild-type RPL22 or RPL22^ha in a control Meox2^+/+:Rpl22^ha/+ mouse, a double heterozygote Meox2^Cre/+:Rpl22^ha/+ mouse, and a Rpl22^ha-expressing homozygous mouse. Blots were probed with anti-RPL22 and anti-HA antibodies. RPL22^ha results in a 23-kDa protein, while the native RPL22 is a 15-kDa protein. (E) Western blot using tissue homogenates from a Rpl22^ha-expressing homozygous mouse. The blot was probed with anti-HA antibody and then reprobed with anti-RPL7 antibody. H, heart; S, spleen; Li, liver; O, ovary; Sk, skeletal muscle; Pa, pancreas; Lu, lung; K, kidney; B, brain. (F) A₂₅₄ absorbance profile of 15% to 50% sucrose density gradients from a Rpl22^ha-expressing homozygous mouse brain in the absence (left) or presence (right) of 200 mM EDTA. Western blot analysis (below each profile) of trichloroacetic acid-precipitated fractions probed with anti-HA antibody.