Generation of NPM-ALK Tg mice.

(A) NPM-ALK cDNA was cloned into a construct containing the CD4 enhancer and promoter as described in “Materials and methods.” (B) Southern blotting of representative animals obtained from different foster mothers. BamHI-digested DNAs were hybridized with a radio-labeled ALK cDNA probe (1 = N1, 2 = N16, 4 = N15, and 8 = N8. Lane 3, 5, 6, and 7 correspond to correspondent normal littermate). (C) The expression and size of the fusion protein was characterized by Western blot. Proteins were extracted from thymi of Tg (N1, N14, and N16) and wild-type (WT) mice and loaded onto SDS-PAGE gel. The expression of the NPM-ALK chimera was detected with polyclonal rabbit anti-ALK antibody. The protein extract from the human ALCL-derived cell line DHL was used as a control. The loading was checked by Western blot for the ubiquitous CDK2 protein. (D) Histology of NPM-ALK Tg mice. Tg thymus (left panels) or spleen (right panels) were fixed in formalin and embedded in paraffin. Hematoxylin and eosin stains (top panels, × 100) showed normal thymus and spleen architecture in the preneoplastic tissue. Immunostaining with anti-ALK antibody (bottom panels) demonstrated a diffuse positivity in Tg thymocytes with stronger signal in medullary lymphocytes (× 100). In Tg spleen the ALK positivity was localized in the periarteriolar T cell areas of the white pulp (× 400). Left insert shows a nuclear and cytoplasmic staining in Tg lymphocytes (× 400); right insert shows a lower magnification of the spleen (× 100).