C57bl 6 tg pf4 icre q3rsko j

Mkl1^–/– mice generated as described previously (73 (link)) were backcrossed onto C57BL/6 and provided by Diane Krause (Yale School of Medicine, New Haven, Connecticut, USA.). Dyrk1a^fl/fl mice on a C57BL/6 background (C57BL/6-Dyrk1a^tm1Jdc/J) were purchased from The Jackson Laboratory. The megakaryocytic deleter strain Pf4^Cre on a C57BL/6 background (C57BL/6-Tg(Pf4-icre)Q3Rsko/J) was purchased from The Jackson Laboratory. Immunodeficient xenotransplant recipients consisted of NOD/SCID IL-2Rγ-null mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, referred to as NSG), purchased from The Jackson Laboratory. PCR genotyping of genomic DNA is described in Supplemental Methods. Procedures included injection of a single i.p. dose of 5-fluorouracil (Teva Pharmaceutical) at 75 mg/kg for 7 days before marrow harvest for ex vivo Mk cultures. Transplantation of human CB Mk into NSG mice followed our recently described approach (15 (link)), as did the quantitation of human circulating platelets and human Mks trapped in the lung. For infusion of platelets, the mice each received 8.5 × 10⁶ platelets harvested from Mk cultures of CB CD34⁺ cells on day 11; as a positive control, a separate cohort of mice were infused with the same number of normal donor platelets.

Reporter mice (Gt(ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo}/J, stock no. 007576) were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). This double fluorescent Cre reporter mouse called mT/mG reporter mouse expresses membrane-targeted tdTomato (′′mT′′) prior to Cre excision and membrane-targeted EGFP (′′mG′′) following Cre excision [22 (link)]. These mice were crossbred with PF4-Cre transgenic mice (The Jackson Laboratory, stock no. 008535, C57BL/6-Tg(Pf4-icre)Q3Rsko/J, Bar Harbor, Maine, USA) to confine the expression of EGFP to megakaryocytes and platelets [19 (link)]. The animals were bred in the animal facility of University Düsseldorf. As WT controls served either littermates, which had a wildtype genotype for mT/mG and C57BL/6 mice, which were bought at Janvier Labs (Le Genest-Saint-Isle, France).
All animal experiments were performed under adherence of the EU Directive 2010/63/EU and German animal welfare act using a protocol approved by the Heinrich-Heine-University Animal Care Committee and he district government of North Rhine-Westphalia (LANUV NRW, permit numbers: 84-02.05.20.12.284, 84-02.05.40.16.073, 84-02.04.2013.A210 and 84 02.04.2016.A493).

Mice were housed and bred under specific pathogen–free conditions at the UCSF Laboratory Animal Research Center. Male and female mice between 8 and 12 weeks of age were used for the experiments. WT C57BL/6J, C57BL/6-Tg(Pf4-icre)Q3Rsko/J, B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTdTomato)Hze}/J, B6.129(Cg)-Gt(ROSA)26Sor^{tm4(ACTB-tdTdTomato,-EGFP)Luo}/J, C57BL/6-Tg(UBC-GFP)30Scha/J, B6.129(Cg)-Lepr^tm2(cre)Rck/J and B6.129S2-CD40lg^tm1lmx/J mice were purchased from The Jackson Laboratory. c-mpl^−/− mice (on a C57BL/6 background) were obtained from Genentech through a material transfer agreement.