Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice
Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice
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DOI：
10.1038/sj.gt.3300689
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发表时间：
1998-07-01
期刊：
影响因子：
5.1
通讯作者：
Watt, FM
中科院分区：
文献类型：
--
作者：
Levy, L;Broad, S;Watt, FM
以前试图在体内实现逆转录病毒转导的人表皮角质形成细胞的长期基因表达的尝试基本上是不成功的。这被不同地归因于未能靶向表皮干细胞、次佳的移植条件或逆转录病毒载体的失活。为了克服这些问题，我们在原代人表皮角质形成细胞中表达了鸡β(1)整合素亚基，这使我们能够在逐个细胞的基础上监测逆转录病毒基因的表达。我们描述了选择高滴度两性包装细胞和感染培养中的角质形成细胞的优化方法。当将转导细胞移植到小鼠体内时，裸鼠和SCID小鼠的移植物存活率相当，但将角质形成细胞与真皮基质结合是必不可少的。使用这些方法，大多数角质形成细胞在移植后至少16周表达鸡β(1)整合素亚基。我们认为，表皮角质形成细胞是一种有吸引力的基因治疗受体细胞。
Previous attempts to achieve long-term gene expression in retrovirally transduced human epidermal keratinocytes in vivo have been largely unsuccessful. This has been variously attributed to a failure to target epidermal stem cells, suboptimal grafting conditions or inactivation of the retroviral vector. In an attempt to overcome these problems we expressed the chick beta(1) integrin subunit in primary human epidermal keratinocytes, which allowed us to monitor retro-viral gene expression on a cell-by-cell basis. We describe optimised methods for selecting high-titre amphotropic packaging cells and for infecting keratinocytes in culture. When transduced cells were grafted into mice, graft survival was comparable in nude and SCID mice, but it was essential to combine the keratinocytes with a dermal substrate. Using these methods the majority of keratinocytes expressed the chick beta(1) integrin subunit for at least 16 weeks after grafting. We conclude that epidermal keratinocytes are attractive recipient cells for gene therapy.