Retrovirus-mediated transduction of cultured epidermal keratinocytes.
Retrovirus-mediated transduction of cultured epidermal keratinocytes.
复制标题
逆转录病毒介导的培养表皮角质形成细胞的转导。
DOI：
10.1111/1523-1747.ep12489019
复制
发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Taichman,LB
中科院分区：
文献类型：
--
作者：
Garlick,JA;Katz,AB;Fenjves,ES;Taichman,LB
逆转录病毒介导的基因转移是在包括角质形成细胞在内的许多细胞类型中引入和表达外源基因的有效手段。然而，角质形成细胞的转导和基因表达的参数尚未进行系统分析。为了进行这样的研究，我们用编码新霉素抗性（neor）和β-半乳糖苷酶（B-gal）基因的逆转录病毒载体转导新生儿包皮细胞培养物。neor基因是显性选择标记，B-gal基因编码组织化学可检测的产物。我们的主要发现如下：1）所有形成集落的角质形成细胞都可以在大于5 × 106集落形成单位/ml的病毒滴度下成功转导：2）转导是通过整合单拷贝逆转录病毒DNA实现的; 3）转导的细胞不处于生长劣势，并且事实上，转导的角质形成细胞的单个克隆可以扩增以产生超过109个细胞，这表明干细胞被转导; 4）尽管大多数转导的集落在高百分比的组成细胞中显示出B-gal染色，但一些集落具有镶嵌或扇形染色模式; 5）与未分化的前体相比，在培养物的分化细胞中，非选择性B-gal基因的表达略高。以高效率扩增干细胞和跟踪克隆后代中转导基因表达的能力将允许在分层上皮组织中进行谱系作图。
Retrovirus-mediated gene transfer is an efficient means of introducing and expressing exogenous gene(s) in many cell types including keratinocytes. However, parameters of transduction and gene expression have not been systematically analyzed for keratinocytes. To carry out such a study we have transduced cultures of newborn foreskin cells with retroviral vectors that encode the genes for neomycin resistance (neor) and for beta-galactosidase (B-gal). The neor gene is a dominant selectable marker and the B-gal gene encodes a histochemically detectable product. Our key findings are the following: 1) all keratinocytes that form colonies can be successfully transduced at a viral titer greater than 5 × 106 colony-forming units/ml; 2) transduction is effected by integration of a single copy of retroviral DNA; 3) transduced cells are not at a growth disadvantage and, in fact, single clones of transduced keratinocytes can be expanded to yield over 109 cells, suggesting that stem cells are transduced; 4) whereas most transduced colonies exhibit B-gal staining in a high percentage of constituent cells, some colonies had a mosaic or sectored staining pattern; 5) expression of the non-selectable B-gal gene was somewhat greater in differentiated cells of the culture as compared to nondifferentiated precursors. The ability to transduce stem cells at a high efficiency and to follow expression of transduced genes in clonal progeny will allow lineage mapping in stratified epithelial tissues.