Demonstration of high-level regeneration of transplantation chimeras by retrovirally transduced cells.

(A) Diagrammatic representation of the integrated Hoxa9 and control proviruses used in this study. The expected sizes of the full-length LTR- and pgk-driven viral transcripts are shown. Restriction sites indicated are KpnI (Kp), EcoRI (E), and NcoI (N). (B) Southern blot analyses of genomic DNA isolated from the bone marrow, spleen, and thymus of Hoxa9and control chimeras. DNA was digested with KpnI to release the integrated Hoxa9-neo (4.1 kb), Hoxa9-EGFP(4.1 kb), neo (2.7 kb), and EGFP (2.7 kb) control viruses (upper panel). The DNA was also digested with eitherEcoRI or NcoI that cuts the integratedHoxa9 and neo or the Hoxa9-EGFP andEGFP proviruses once, respectively, thus generating a unique fragment for each proviral integration site (lower panel). The membranes were hybridized with a neo probe to detect theHoxa9-neo and neo proviruses and anEGFP probe to detect Hoxa9-EGFP andEGFP proviruses. (C) Northern blot analysis of total RNA (10 μg) isolated from bone marrow, thymus, and/or spleen of some of the mice described in panel B. The Hoxa9 probe detects the expected viral LTR-driven Hoxa9 messages and theneo probe both the LTR- and pgk-driven viral messages, as depicted in panel A. B indicates bone marrow; S, spleen; T, thymus.