Design, expression, and functionality of HOX swap constructs. (A) BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) alignment of human HOXA1 and HOXA9 proteins. Amino acid sequences are given in the 1-letter code. Identical residues are in bold type. The tryptophan essential for interaction with PBX cofactors is marked in red. The amino-terminal basic extension and the core homeodomain, including recognition helix 3, are highlighted. (B) Left panel: 3-dimensional structure of a murine Hoxa9/Pbx1 dimer bound to DNA (protein structure database accession: 1PUF). Only part of Hoxa9 and the DNA helix are shown. For the N-terminal extension upstream of the core homeodomain, the respective amino acids are given in the 1-letter code. Green represents DNA, and amino acids are colored according to their charge: blue represents basic; yellow, hydrophobic; and red, acidic. This figure is a screenshot of a visualization done with the software Cn3D (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml). Top right panel: Junction sequences of the swap clones with their respective HOXA1 and HOXA9 contributions. Bottom right panel: Immunoblot of chimeric HOX proteins and their parental counterparts. Nuclear extracts were analyzed from Phoenix-E packaging cells transfected with the respective retroviral plasmids. After separation by SDS-PAGE, proteins were blotted and detected by an anti-HA tag antibody. (C) Colony counts obtained in replating assays. Primary, hematopoietic precursors were transduced with the constructs as indicated and replated twice in cytokine containing methocel media. Colony numbers are given as mean ± SD of 3 biologic replicates.