Selective expression of Cre in megakaryocytes and platelets in Pf4-Cre transgenic mic. (A) Pf4-Cre transgene construct. A codon-improved cDNA for Cre-recombinase was inserted by homologous recombination into bacterial artificial chromosome DNA containing the mouse Pf4 gene. Numbered boxes indicate Pf4 exons; polyA, bovine growth hormone polyadenylation signal; RV, EcoRV restriction sites. Note that exon 1 of Pf4 is deleted in the transgene construct. (B) Quantification of Cre mRNA expression by real-time PCR in Pf4-Cre mice. RNA from unfractionated bone marrow (BM), bone marrow cells separated into CD41⁺ or CD41⁻ fractions, and platelets (PLT) were analyzed. Relative expression levels calculated by the ΔΔCT method are shown. One bone marrow sample was set to the value of 1. Horizontal lines mark the mean of the values from 3 mice. (C) Expression of the Cre protein in tissues from Pf4-Cre mice. Western blot probed with an anti-Cre antibody (top) and reprobed with an anti–β-actin antibody (bottom) is shown. SPL indicates, spleen; THY, thymus; BM, bone marrow; BRA, brain. (D) Detection of Cre-mediated recombination in Pf4-Cre mice using the ROSA26-lacZ reporter. Bone marrow cytospins from Pf4-Cre transgenic mice and controls are shown. Excision of a stop cassette flanked by loxP sites results in β-galactosidase activity that can be visualized by X-Gal staining (blue color). The samples were counterstained with nuclear fast red to visualize nuclei. Note that the blue staining is limited to cells with megakaryocyte morphology (arrows). (E) CD41⁺ magnetic cell sorting (MACS)–purified megakaryocytes from Pf4-Cre × ROSA26-lacZ mice were stained for acetylcholine esterase (left) or β-galactosidase activity (right).