Insertion of a potent antisickling human β-globin gene into a lentiviral vector. (A) Schematic of the human β-globin LCR and downstream genes. HS cores are indicated by arrows. Genes are illustrated by filled boxes and are represented according to scale. (B) The lentiviral/antisickling β-globin expression construct is shown. HS2 (1203 bp), HS3 (1213 bp), and HS4 (954 bp) sequences, the 3′ globin enhancer, the 266-bp β-globin promoter (βp) and the β^AS3 globin gene are drawn to scale. The HIV-1 LTR is shown with a 3′ SIN deletion; ψ indicates packaging signal; SD and SA, splice donor and acceptor sites, respectively; RRE, Rev-responsive element; cPPT/CTS, central polypurine tract or DNA flap/central termination sequence; and WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (C) Lentiviral vector copy number determination. Lane 1 is the PCR product obtained from bone marrow of a KO-TG sickle mouse after amplification, labeled primer extension, and Bsu36I digestion; lanes 2, 3, and 4 are products of representative lenti/β^AS3-transduced mice; and lane 5 is a knockout-transgenic animal expressing exclusively human HbAS3.