|
|
Blood, Vol. 108, Issue 8, 2545-2553, October 15, 2006
Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity
Blood Modlich et al.
108: 2545
Supplemental materials for: Modlich et al
32D cells, an Interleukin-3 (IL-3) dependent murine myeloid cell line, were cultured in RPMI (PAA, Cölbe, Germany) supplemented with 10% fetal calf serum (FCS) and 2ng/ml murine IL-3. Cells were split in regular intervals to maintain densities <106/ml. Equal infectivity of LTR and SIN vector stocks with identical titers was assessed by transducing 32D cells with increasing MOI and subsequent flow cytometry (Fig. S1), determination of copy number by Southern blot (Fig. S2), and quantitative PCR detecting the vectors’ PRE sequences in unselected cell populations (Table S1). A standard curve for the PCR was derived using a clone with a known number of insertions as determined by Southern blot. Despite inter-experimental variations, the side-by-side comparison of LTR and SIN vectors did not reveal a significantly superior infectivity of either of the two vector types. Intra-experimental differences between the two types of vectors were small, except for the first comparison of LTR and SIN vectors at high MOI where day 3 transgene copy number of the SIN vector was only two thirds of the LTR value, and day 7 transgene copy number of the SIN vector even lower. However, PCR determinations of vector copy numbers may be complicated by several factors, including persistence of unintegrated retroviral DNA early after transduction.
Files in this Data Supplement:
|
|
