Expression of an Mpl transgene (Yall) in megakaryocytes. (A) Yall transgene construct: a 2-kb fragment corresponding to the genomic sequence immediately 5′ of the Mpl ATG start codon was placed as a promoter in front of the mouse Mpl cDNA. An alignment of endogenous mouse Mpl genomic sequence (above) and of the transgene (below) in the region upstream of the ATG start codon is shown. An SV40-derived sequence element containing an intron was placed at the 3′ end of the construct to ensure polyadenylation of the transcript. (B) Specific expression of the Yall transgene in bone marrow megakaryocytes. Expression of transgenic (■) and endogenous Mpl mRNA () was measured by qPCR in mouse organs from Yall;Mpl^−/+ mice (error bars represent SD). Bone marrow was further fractionated into CD41⁺ and CD41⁻ cells. BM indicates whole bone marrow; SPL, spleen; THY, thymus; LIV, liver; KID, kidney; LUN, lung; HEA, heart; INT, intestine; BRA, brain; OVA, ovary; and TES, testis. Primers for qPCR were specific for transgene and endogenous Mpl mRNA, respectively, and control cDNA synthesis reactions without reverse transcriptase were analyzed to exclude amplification of genomic DNA. Expression of mouse Rpl19 was used for normalization and relative expression was calculated with the ΔΔCT method using one bone marrow sample as calibrator. The mean value of 3 mice is shown. (C) Western blot with protein extracts from magnetic-activated cell sorter (MACS)–isolated megakaryocytes probed with antibodies against Mpl protein and the megakaryocyte-specific glycoprotein V (GP V). Megakaryocytes from wild-type (wt) mice and nontransgenic (−) or transgenic (Yall) Mpl^−/− mice were analyzed.