FIG. 1.
Gene targeting of exon 3 in the Mpl gene. (A) Schematic of the targeting strategy. The 5′ loxP site was inserted upstream of exon 3, followed by the insertion of an Frt-pGK-loxP-Neo-Frt-loxP selection cassette. The expected EcorV-digested fragments for wild-type and targeted alleles are indicated by large arrows. The 5′ external probe is indicated by a short line and the 5′ and 3′ homology arms are shown in gray. LoxP and Frt sites are shown by filled triangles and empty triangles, respectively. Genotyping primers are shown as small arrows. Flp, Flp recombinase mice; PF4cre, Platelet factor 4 cre recombinase mice. (B) Southern blot analysis with the 5′ probe. 1–5, Mplf/+ mouse lines; HYB, C57BL/6/129 hybrid; B6, C57BL/6; 129, 129/SvEv mice. (C) PCR analysis of mouse tail DNA obtained from Mplf/+ intercrosses, indicating transmission of the targeted (KO) allele. (D) qPCR of cDNA from MK showing expression of Mpl, n = 3–5 mice per genotype, Student's t-test, *P < 0.001. cDNA, complementary DNA; KO, knockout; MK, megakaryocytes; qPCR, quantitative polymerase chain reaction.