Lei Pedro, Andreadis Stelios T
Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, NY, USA.
Methods Mol Biol. 2008;433:367-79. doi: 10.1007/978-1-59745-237-3_22.
Skin is an attractive target for gene modification to treat skin diseases, wound healing, or even systemic disorders. Although retroviral transduction results in permanent genetic modification, differentiation and eventually loss of the transduced cells from the epidermis and in temporary transgene expression. Therefore, it is important to develop methods that promote gene transfer to epidermal stem cells, which self-renew and regenerate the epidermis for extended periods of time. Here we describe an efficient protocol that results in high levels of retroviral gene transfer to human epidermal stem cells by immobilizing retrovirus on a recombinant fibronectin (rFN) fragment. In contrast to the traditional method, transduction on rFN promotes gene transfer to epidermal stem cells and prevents loss of clonogenic potential due to exposure of cells to retroviral supernatant. Notably, transduction on rFN does not require addition of toxic polycations such as polybrene. Overall this method provides a simple, fast, and efficient means to modify human epidermal stem cells for cutaneous gene therapy and for biological studies that require stable genetic modification.
皮肤是基因改造的一个有吸引力的靶点，可用于治疗皮肤疾病、伤口愈合，甚至全身性疾病。尽管逆转录病毒转导可导致永久性基因改造，但转导的细胞会从表皮分化并最终丢失，且转基因表达是暂时的。因此，开发促进基因转移至表皮干细胞的方法很重要，表皮干细胞可长时间自我更新并再生表皮。在此，我们描述了一种高效方案，通过将逆转录病毒固定在重组纤连蛋白（rFN）片段上，实现向人表皮干细胞的高水平逆转录病毒基因转移。与传统方法相比，在rFN上进行转导可促进基因转移至表皮干细胞，并防止由于细胞暴露于逆转录病毒上清液而导致克隆潜能丧失。值得注意的是，在rFN上进行转导不需要添加有毒的聚阳离子，如聚凝胺。总体而言，该方法提供了一种简单、快速且高效的手段，用于改造人表皮干细胞以进行皮肤基因治疗以及用于需要稳定基因改造的生物学研究。
