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Skoda; Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo. Blood ; 4 : — To generate transgenic mice that express Cre-recombinase exclusively in the megakaryocytic lineage, we modified a mouse bacterial artificial chromosome BAC clone by homologous recombination and replaced the first exon of the platelet factor 4 Pf4also called CXCL4with a codon-improved Cre cDNA.
Several strains expressing the transgene were obtained and one strain, Q3, was studied in detail. The Pf4-Cre transgenic strain will be a valuable tool to study megakaryopoiesis, platelet formation, and platelet function. Deletion of ubiquitously expressed genes often results in severe phenotypes that interfere with the analysis of particular cell types or tissues.
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To achieve specific Cre expression, we used a bacterial artificial chromosome BAC clone containing the entire mouse Pf4 promoter. The Pf4-Cre transgene Article Summary of Kreppner was generated by homologous recombination in bacteria for details, see legend to Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article. Reverse transcription was performed with Omniscript reverse transcriptase Qiagen, Hilden, Germany. Platelets were obtained from platelet-rich plasma by centrifugation at g for 5 minutes.
Purity was assessed on an Advia hematology analyzer Bayer, Leverkusen, Germany.
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Leukocytes were purified from buffy coat by red cell lysis. Images were acquired using Leica FireCam software version 1. No fixatives were used. Megakaryocytes were Article Summary of Kreppner in hypotonic citrate buffer prior to FACS analysis. A Cre -recombinase cDNA optimized for codon-usage in eukaryotic cells and containing a nuclear localization signal and a myc tag was inserted by homologous recombination in bacteria into an approximately kb mouse BAC clone containing the Pf4 gene Figure 1 A; for details, see Figure S1.
Five of the 6 transgenic strains expressed the transgene, but only 2 strains showed a restricted expression pattern Table S1.
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One of these strains, Q3, with a single copy transgene was selected for detailed analysis. 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. Note that exon 1 of Pf4 is deleted in the Krepppner construct. One bone marrow Kreppnrr 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. Bone marrow cytospins from Pf4-Cre transgenic Article Summary of Kreppner and controls are shown. The samples were counterstained with nuclear fast red to visualize nuclei. Note that the blue staining is limited to cells with megakaryocyte morphology arrows.
To demonstrate that the Cre protein is functional and can mediate lineage-specific excision, we crossed the Q3 strain with ROSAlacZ reporter mice. Even though large differences in lacZ staining intensities between individual megakaryocytes were noted, the majority of megakaryocytes displayed at least small amounts of blue staining.
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This finding was confirmed by LacZ staining of cytospin slides with more than a hundred purified megakaryocytes Figure 1 E. Cre activity was absent from all other inspected organs data not shown. Thus, Cre expression and functional activity follows the lineage distribution expected for the PF4 promoter.
Numbered black boxes represent coding exons, loxP sites are depicted as gray triangles.]
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