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. 2009 Mar 10;106(13):5192–5197. doi: 10.1073/pnas.0811712106

Fig. 1.

Fig. 1.

Generation of p57p27KI mice. (A) Structures of p27 and p57. The 2 proteins share conserved CKI and QT domains in their NH2- and COOH-terminal regions, respectively. Mouse p57 possesses a unique central domain that is not conserved in p27 or in human p57. (B) Schematic representations of the wild-type mouse p57 allele (p57WT), the targeting vector, the knocked-in p27 allele with a LoxP-neo cassette (p57p27KI-neo), and the knocked-in p27 allele after removal of the neo cassette by Cre recombinase. The expected sizes of DNA fragments that hybridize with the indicated probe in Southern blot analysis are shown. The ORF for mouse p27 tagged at its NH2 terminus with HA is indicated by the red box. All introns of the p57 gene were removed in the resulting targeted allele to avoid splicing defects. E1, EcoRI; N, NotI; Nd, NdeI. Indicated are the initiation (1 asterisk) and stop (2 asterisks) codons of the p57 gene. (C) Southern blot analysis of EcoRI-digested DNA from ES cells harboring the indicated alleles. The sizes of the hybridizing fragments correspond to those indicated in B. (D) Immunoblot and PCR analyses of the expression and imprinting of the knocked-in p27 allele. Kidneys of newborn mice [postnatal day (P)0] were lysed and subjected to immunoblot analysis with antibodies to p57, to HA, and to p27. The asterisk indicates nonspecific bands, and the band positions for endogenous (Endo.) and knocked-in (KI) p27 are shown. Genomic DNA of the corresponding animals was subjected to PCR analysis to determine their genotypes. Brackets indicate a paternal imprinted allele. Both p57 and HA-p27 were expressed only from the maternal allele. WT and KI indicate the wild-type and knocked-in alleles, respectively. (E) Immunohistochemical analysis of the indicated tissues of wild-type (p57WT) or p57p27KI neonates performed with antibodies to p57 or to HA. Sections were counterstained with hematoxylin. Arrowheads indicate the same types of cells expressing either p57 or HA-p27 in p57WT and p57p27KI mice, respectively. R, retina; L, lens; P, zone of proliferative cells; F, zone of flattened cells; H, zone of hypertrophic cells. (Scale bar, 50 μm.)