Effect of HS1 on transgene expression. (Ai) Schematic representation of vector pairs used to evaluate the effect of 5′HS1 element on transgene expression. The human β-globin transgene (gene, promoter, and 3′enhancer) is represented in red. The locus control region (LCR) elements HS2 and HS3 are indicated in green and HS4 in blue. The orange box indicates the HS1 element. The triangle above the 3′LTR indicates deletion in the U3 region making it a self-inactivating (SIN) vector. Numbers between vector pairs indicate the size of promoter used in each vector. The letters “S” and “T” in vector names indicates 265bp and 615bp promoters, respectively. Number “9” in the vector name indicates LCR2-3-4, while “10” stands for LCR1-2-3-4. Through the course of the study, none of the vectors expressed transgene in mouse lymphoma (EL4) cells or undifferentiated MEL cells (data not shown), confirming tissue and differentiation stage specificity of the vectors. (Aii) Quantification of vector expression in independent MEL cell pools. Expression at the RNA level (Huβ/(Huβ+Muβ)) is normalized to vector copy number (VCN). P values were calculated using Student t test. The n values indicate the number of independent MEL cell pools. (B) Long-term stability of vector copy number in vivo assayed by TaqMan analysis. Three sample mice for each vector are shown. (C) Human β-globin transgene mRNA expression in peripheral blood (PB) shown as fraction of total β-globin mRNA and normalized to vector copy [(Huβ/(Huβ + Muβ)/VCN]. For each vector, bars indicate time points during the experiment, in order from left: Week 6, 12, 17, 23, 29, and 37. The number of mice ranged between 8 and 19 per group (total: 73 mice). P values were calculated using Student t test. Error bars in panels Aii and C are SD. (D) Cellulose acetate gel electrophoresis shows chimeric Hb (cHb, mα_2:hβ₂) levels in vector-transduced bone marrow chimeras. Data shown for 3 representative animals from each group at week 23 after transplantation. Vertical lines have been inserted to indicate repositioned gel lanes. Results were very similar at different time points. Control lanes contain normal C57BL/6 (B6) or Hbb^Th3/+ (Th) blood samples. The fraction of chimeric Hb (%cHb) relative to total hemoglobin (cHb/cHb+mHb) and vector copy number (VCN) are indicated below each sample.