Southern analysis of HIV antisense transcription from vCWRHIVASVN in transduced marrow and cord blood-derived hematopoietic progenitor cells after RT-PCR amplification. (A) RNA was extracted from week-5 to -7 LTBMCs and antisense sequences were reverse transcribed and amplified using primers 1a and 1b. The amplified products were resolved on a 1.2% agarose gel, transferred to nitrocellulose, and hybridized with an RSV LTR and antisense-specific probe. HIIC21, containing 1 copy of integrated vector per cell, served as the positive control. The 530-bp antisense transcript-specific product is shown. +RT and −RT refer to the presence or absence, respectively, of reverse transcription before amplification. The absence of signals in these lanes indicates that the antisense signals were RNA-specific. ψ, RT-PCR analysis of cells (donor 20) exposed to a sham stock of vector prepared in the absence of AAV rep and cap genes. Untransduced (Untd) cells from each donor tested served as negative controls. Td, transduced cultures. (B) HIV LTR antisense transcription in LTC-ICs initiated with CD34⁺CD38⁻ cord blood cells 8 weeks after transduction. A representative sample, CB6, was transduced with vCWRHIVAPAP at MOI of 3 (particle MOI, 600) and vCWRHIVASVN at MOI of 0.1 (particle MOI, 50). Cells were harvested from 6 week LTBMCs, washed, and placed in colony-forming assays. RNA was extracted from colonies after 3 weeks, reverse transcribed, and amplified with primers 1a and 1b. The 530-bp antisense product was evident only after reverse transcription.