Generation of TVA-expressing transgenic mice.

(A) Schematic of the transgene cassette used to express TVA, indicating the position of the 5′ (−783) and 3′ (+32) ends of the humanGPIIb regulatory sequence ligated to the 950 bptv-a cDNA. Numbering is assigned relative to 0 for the native GPIIb transcription start site. The horizontal arrow indicates the transcribed message, and translation begins with a methionine at the 5′ end of the tv-a cDNA. Arrowheads indicate the positions of primers used for RT-PCR. (B) A founder mouse (9552) positive for the transgene by Southern blot was bred with nontransgenic littermates. RNA isolated from the bone marrow of a transgene-containing offspring (+) and a nontransgenic littermate (−) was subjected to RT-PCR without (−) and with (+) reverse transcriptase in the reaction. The 420-bp band indicative of TVA mRNA (arrow) is only seen when reverse transcriptase is used (lane 1 vs lane 2). Bone marrow from mice lacking the transgene does not express TVA mRNA (lanes 3, 4). DNA size markers (bp) are indicated on the left. (C) Hematoxylin and eosin stain of bone marrow from a TVA-expressing mouse demonstrates megakaryocytes (left panel). Immunohistochemical staining demonstrates TVA expression on megakaryocytes in the bone marrow of TVA-expressing 9552 offspring (center panel) but no TVA expression in bone marrow from nontransgenic littermates (right panel). (D) Independent FACS analysis using isotype controls plotted against forward scatter was used to identify a generous gate that encompasses all cells stained by either anti-CD41 or anti-TVA antibody. Two-dimensional FACS analysis using anti-CD41 and anti-TVA antibodies demonstrates dual staining of essentially all gated cells.