Stockschlaeder M A, Storb R, Osborne W R, Miller A D
Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Hum Gene Ther. 1991 Spring;2(1):33-9. doi: 10.1089/hum.1991.2.1-33.
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.
携带新霉素磷酸转移酶（neo）基因的逆转录病毒载体已被证明能以相对较高的频率赋予犬角质形成细胞G418抗性。为了研究角质形成细胞作为基因治疗潜在靶细胞的实用性，我们使用了一种包含人腺苷脱氨酶（hADA）和neo基因的逆转录病毒载体（LASN）。我们在此表明，经LASN转导的犬角质形成细胞表达高水平的hADA，这是一种具有治疗相关性的人类蛋白质。在含有G418的培养基中选择经LASN转导的角质形成细胞，得到了一群表达更高水平hADA的细胞，其表达量比内源性犬ADA水平高约80倍。然而，经G418选择的细胞增殖潜力降低，形态改变，表明发生了终末分化。为了测试L-组氨醇对角质形成细胞选择是否比G418更有益，我们构建了两种包含neo和组氨醇脱氢酶（hisD）基因的逆转录病毒载体。原代角质形成细胞与产生这些载体的经致死性照射的PA317逆转录病毒包装细胞共培养，在G418或L-组氨醇中产生了12% - 53%的耐药菌落。与G418不同，在L-组氨醇中选择转导的角质形成细胞对含有载体的耐药细胞的增殖潜力或形态没有明显影响。鉴于这种选择系统的实用性，构建了两种基于hisD的通用构建体，它们含有用于从逆转录病毒启动子或内部人巨细胞病毒立即早期启动子进行cDNA表达的克隆位点。我们的结果表明，hisD将是用于角质形成细胞分化研究以及为基因治疗目的将基因转移到角质形成细胞中的有用选择标记。
