Fig. 2.
Fig. 2. Amplification of vCWRHIVAPAP sequences from individual LTC-IC colonies. CD34 cells were transduced with vCWRHIVAPAP at a functional MOI of 3 (particle MOI, 600) on day 0 and plated in long-term culture as described in Materials and Methods. Cells were harvested from stromal layers at designated time points, washed, and plated in methylcellulose with no G418 selection and colonies were plucked after 2 weeks. DNA was extracted and amplified for either the AAV vector using primers RPSP and DLASP or for β globin. β Globin served as a control for template integrity. The 418 HIVA band denotes the vector signal. The 268-bp Glo band denotes the β globin signal. Representative colonies from untransduced controls (Untd) are shown. Copy number controls are included in each analysis. For HIVA, the copy number corresponds to 12, 120, and 1,200 copies of the genome. For β globin, copy number controls show amplification from 80, 160, and 1,000 cells. (A) Week-5 LTC-ICs from donor TS. All colonies except one had intact DNA, albeit at varying quantities. The absence of a β globin signal from colony 9 indicated that the DNA template was inadequate. (B) Amplification of vCWRHIVAPAP sequences from individual week-8 LTC-IC colonies from donor TS. All colonies analyzed had intact DNA templates and 9 of 15 showed vector-specific signals. The standard curve is reversed in this experiment. (C) Amplification of vCWRHIVAPAP sequences of individual week-8 (transduced lanes 1 through 10) and week-10 (transduced lanes 11 through 20) LTC-IC colonies from donor HJ. Nine of 10 week-8 LTC-ICs had intact templates. Six of these showed vector-specific signals. All 10 week-10 colonies analyzed had intact DNA, and 6 of these were transduced. (D) Amplification of vCWRHIVAPAP sequences of individual week-5 and -10 LTC-IC colonies from donor FK. Colony 4 had intact albeit very low amounts of DNA as evidenced by a faint β globin signal on a prolonged radioautographic exposure. (E) Amplification of methylcellulose samples from between colonies in transduced LTC-IC wells. Ten separate samples of methylcellulose were amplified with primers RPSP and DLASP using PCR conditions identical to above. NT, no template; C, CWRHIVAPAP control (10 copies). This film was exposed for 36 hours longer than exposures in (A) through (D)

Amplification of vCWRHIVAPAP sequences from individual LTC-IC colonies. CD34 cells were transduced with vCWRHIVAPAP at a functional MOI of 3 (particle MOI, 600) on day 0 and plated in long-term culture as described in Materials and Methods. Cells were harvested from stromal layers at designated time points, washed, and plated in methylcellulose with no G418 selection and colonies were plucked after 2 weeks. DNA was extracted and amplified for either the AAV vector using primers RPSP and DLASP or for β globin. β Globin served as a control for template integrity. The 418 HIVA band denotes the vector signal. The 268-bp Glo band denotes the β globin signal. Representative colonies from untransduced controls (Untd) are shown. Copy number controls are included in each analysis. For HIVA, the copy number corresponds to 12, 120, and 1,200 copies of the genome. For β globin, copy number controls show amplification from 80, 160, and 1,000 cells. (A) Week-5 LTC-ICs from donor TS. All colonies except one had intact DNA, albeit at varying quantities. The absence of a β globin signal from colony 9 indicated that the DNA template was inadequate. (B) Amplification of vCWRHIVAPAP sequences from individual week-8 LTC-IC colonies from donor TS. All colonies analyzed had intact DNA templates and 9 of 15 showed vector-specific signals. The standard curve is reversed in this experiment. (C) Amplification of vCWRHIVAPAP sequences of individual week-8 (transduced lanes 1 through 10) and week-10 (transduced lanes 11 through 20) LTC-IC colonies from donor HJ. Nine of 10 week-8 LTC-ICs had intact templates. Six of these showed vector-specific signals. All 10 week-10 colonies analyzed had intact DNA, and 6 of these were transduced. (D) Amplification of vCWRHIVAPAP sequences of individual week-5 and -10 LTC-IC colonies from donor FK. Colony 4 had intact albeit very low amounts of DNA as evidenced by a faint β globin signal on a prolonged radioautographic exposure. (E) Amplification of methylcellulose samples from between colonies in transduced LTC-IC wells. Ten separate samples of methylcellulose were amplified with primers RPSP and DLASP using PCR conditions identical to above. NT, no template; C, CWRHIVAPAP control (10 copies). This film was exposed for 36 hours longer than exposures in (A) through (D)

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