l-Histidinol Provides Effective Selection of Retrovirus-Vector-Transduced Keratinocytes Without Impairing Their Proliferative Potential

Abstract

Retroviral vectors carrying the neomycin phosphotransferase (neo) gene have been shown to confer G418 resistance to canine keratinocytes at relatively high frequency. To investigate the usefulness of keratinocytes as potential target cells for gene therapy, we used a retroviral vector (LASN) that contains both human adenosine deaminase (hADA) and neo genes. We show here that LASN-transduced canine keratinocytes expressed high levels of hADA, a human protein of therapeutic relevance. Selection of LASN-transduced keratinocytes in medium containing G418 resulted in a population of cells that expressed even higher levels of hADA, about 80-fold higher than the endogenous canine ADA level. However, the G418-selected cells had a reduced proliferative potential and altered morphology indicative of terminal differentiation. To test whether L-histidinol is more beneficial for selection of keratinocytes than G418, we constructed two retroviral vectors that contain both the neo and the histidinol dehydrogenase (hisD) genes. Cocultivation of primary keratinocytes with lethally irradiated PA317 retrovirus packaging cells that produce these vectors gave rise to 12-53% drug-resistant colonies in either G418 or L-histidinol. In contrast to G418, selection of transduced keratinocytes in L-histidinol had no apparent effect on the proliferative potential or morphology of drug-resistant cells containing the vectors. Given the utility of this selection system, two hisD-based generic constructs containing cloning sites for cDNA expression from either the retroviral promoter or from an internal human cytomegalovirus immediate early promoter were constructed. Our results suggest that hisD will be a useful selectable marker for use in studies of keratinocyte differentiation and for transfer of genes into keratinocytes for the purposes of gene therapy.

Phosphorylation of Tyrosine 720 in the Platelet-Derived Growth Factor α Receptor Is Required for Binding of Grb2 and SHP-2 but Not for Activation of Ras or Cell Proliferation

“…They were maintained in Dulbecco's modified Eagle's (DME) medium supplemented with 10% fetal bovine serum (FBS). The wild-type (WT) and F720 human ␣PDGFR cDNAs were subcloned into the pLXSHD retroviral vector (57) and expressed in PhB cells, as previously described (27). Briefly, DNA was transfected into ⌿2 cells, and the viral supernatant from these cells was used to infect PA317 cells.…”

ZPK inhibits PKA induced transcriptional activation by CREB and blocks retinoic acid induced neuronal differentiation

“…Maloney murine leukemia virus-based retroviral vector pLXSN (gift from Dr Osborne's Lab (Stockschlaeder et al, 1991)) was used to express the full length ZPK. ZPK cDNA was cloned into the EcoRI site of pLXSN.…”

Differentiation-specific enhancer activity in transduced keratinocytes: a model for epidermal gene therapy

“…This enabled relatively low viral titre vectors to be tested in an in vivo system by circumventing the problems of stem cell transduction and/or G418-induced terminal senescence 4,16 observed with primary foreskin-derived keratinocytes.…”

“…4 Here we have investigated the effect that the different keratinocyte-specific promoter-proximal elements exerted in vivo after transduced keratinocytes were transplanted to nude mice. The HaCaT keratinocyte cell line 15 was used as a model to overcome the problems associated with transduction/G418 selection 16 of normal human keratinocytes and the grafting procedure was modified to prolong the survival time of transplanted keratinocytes.…”