Page S M, Brownlee G G
Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, UK.
Gene Ther. 1998 Mar;5(3):394-402. doi: 10.1038/sj.gt.3300591.
HaCaT cells, a spontaneously immortalised, nontumorigenic keratinocyte line, were used as a more amenable model than primary keratinocytes for ex vivo-mediated gene transfer. These cells were transduced with retroviral vectors containing the factor IX cDNA under the control of a cytomegaloviral (CMV) promoter/enhancer alone or as hybrids with either the human papilloma virus-16 (HPV-16), keratin 14 (hK14) or keratin 5 (hK5) regulatory elements. Unlike primary keratinocytes, HaCaT cells tolerated transduction and G418 selection well. The HPV-16 and hK5 hybrid constructs were disproportionately more active in primary keratinocytes than in the basal-like HaCaT cells. After skin grafting to athymic mice, transduced HaCaT cells differentiated to form a stratified epidermis that remained viable for at least 99 days in some mice. Factor IX in plasma of mice grafted with vectors containing the HPV-16 and hK5 elements was two- to three-fold higher than with vectors containing the CMV promoter alone. These results are consistent with the expected up-regulation in differentiated suprabasal cells by the HPV-16 and hK5 elements. Enhancers may be useful in specifically targeting the differentiated layer of the epidermis or achieving higher levels of gene expression after transplantation.
HaCaT细胞是一种自发永生化的非致瘤性角质形成细胞系，与原代角质形成细胞相比，它是一种更适合用于体外介导基因转移的模型。这些细胞用逆转录病毒载体转导，该载体单独包含在巨细胞病毒（CMV）启动子/增强子控制下的因子IX cDNA，或者作为与人乳头瘤病毒16型（HPV-16）、角蛋白14（hK14）或角蛋白5（hK5）调控元件的杂种。与原代角质形成细胞不同，HaCaT细胞对转导和G418筛选耐受性良好。HPV-16和hK5杂种构建体在原代角质形成细胞中的活性比在基底样HaCaT细胞中高得多。将转导后的HaCaT细胞移植到无胸腺小鼠后，其分化形成分层表皮，在一些小鼠中至少存活99天。移植含HPV-16和hK5元件载体的小鼠血浆中的因子IX比单独含CMV启动子载体的小鼠高两到三倍。这些结果与HPV-16和hK5元件在分化的基底上层细胞中预期的上调一致。增强子可能有助于特异性靶向表皮的分化层或在移植后实现更高水平的基因表达。
