PMID: 8952153Sep 1, 1996Paper

beta-galactosidase transduced T lymphocytes: a comparison between stimulation by either PHA and IL-2 or a mixed lymphocyte reaction

Haematologica
M Di IanniA Tabilio

Abstract

Retroviral-mediated gene transfer stably introduces exogenous genes into normal and neoplastic cells of the hematopoietic system. We used two retroviral vectors [the first, FLac, expresses a chimeric protein (Sh-ble::LacZ) between the product of the phleomycin resistance gene (Sh-ble) and the bacterial beta-galactosidase encoded by the LacZ gene; the second, NuNL vector, contains a fusion sequence (LacZ::Neo) that expresses the LacZ and the neomycin resistance genes] to transduce T lymphocytes derived from the peripheral blood of healthy human donors. Two lymphocyte activation procedures were employed: a) phytohemagglutinin/interleukin-2 (PHA/IL-2) polyclonal stimulation; b) allogeneic stimulation in a mixed irradiated or non irradiated lymphocyte reaction, both supplemented with IL-2 (MLR/IL-2). Infection was achieved by co-cultivating activated T cells with the producing amphotropic cell line pretreated with mitomycin C for 96 hours. Infection and transduction efficiency were assayed by LacZ gene expression, which is detected as indigo blue staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X..Gal). The highest percentage of transduced T cells was reached on the 3rd PHA/IL-2 and on 9t...Continue Reading

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