Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38

Abstract

Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%+/-8.5% and the mean cell viability 74%+/-17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells.

Figure 1.

Successful transduction of CLL cells with lentivirus. (A) Treatment of CLL cells with lentivirus containing the genetic codes for CD38 (i), GFP (ii), and a truncated rat CD2 (iii) resulted in high levels of transduction (87%, 70% and 43%, respectively). (B) CLL cells from a CD38 negative patient (i) were infected with a CD38 lentivirus and 94% transduction was achieved (ii). Infection with a GFP control lentivirus saw a 9.7% increase in CD38 expression (iii).

Figure 2.

Expression of CD38 on virally infected CLL cells. A dose dependent increase in CD38 expression was observed in both the number of cells expressing CD38 (A) and in the MFI (B) of samples following treatment with CD38 virus (diamonds). No such increase is observed following the addition of a comparable amount of GFP virus (squares). Following transduction a high level of CD38 expression was observed on the surface of CLL cells following 24 h incubation. This level was sustained over a period of five days as measured by the percentage of cells expressing the CD38 antigen (C) and by the MFI (D) of the sample.

Figure 3.

Expression of CD38 in multiple patient samples. (A) The mean expression of CD38 in the untreated samples was 3±1.8% (n=17). Following treatment with CD38 virus a mean of 87±8.5% (n=17) of CLL cells expressed the CD38 antigen. A mean of 8±5.8% CD38 expression (n=7) was observed in samples treated with control GFP virus. (B) Morphology of untreated and lentivirus treated CLL cells following 48 h incubation. Pictures were taken using a Zeiss Axio microscope equipped with a digital camera following Giemsa staining (×100 magnification).