In contrast, lentiviral (LV) vector capsids have a larger physical size (about 100 nm) and are capable of packaging promoter/transgene sequences over twice that of AAV [9]. This property is invaluable for transfer of large promoter constructs or transgene coding sequences which cannot be accommodated within AAV vectors [2,10,11]. Furthermore, concentration and purification of LV vectors may be accomplished by ultracentrifugation alone, whereas AAV vectors require the use of column chromatography to generate pure high titer preparations.
High Pack Ivan Gorokhov
As an alternative to removing the CMV/LTR from the backbone vector, a sequence that is necessary for producing high titer viruses in the packaging cell line, we tested the effectiveness of a synthetic TB element inserted immediately upstream of the MCS (Figure 1). The TB element has been previously characterized as containing a synthetic poly-A (SPA) sequence (AATAAA sequence and a GT/T-rich sequence with the correct spacing of 22-23 nucleotides between them) [22], and a C2 transcriptional pause site [23], which slows the processivity of the RNA polymerase complex. Quantitative assessment of TB function by both flow cytometry (Figure 2A) and quantitative RT-PCR (Figure 2B) indicated a reduction in eGFP expression by approximately 85%. A second TB element arranged in tandem did not enhance the blocking effect (data not shown). Moreover, the mean fluorescence intensity (MFI) in the flow cytometry samples (Figure 2) indicated a reduction in the amount of eGFP protein produced in the small population of transfected cells that circumvented the transcriptional block. Cells transfected with pFMGW had a MFI of 34.32, while inclusion of the TB element in pFTMGW reduced the MFI to 8.3. 2ff7e9595c
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