Sat E, Leung K H, Bruckner-Tuderman L, Cheah K S
Department of Biochemistry, The University of Hong Kong, China.
Gene Ther. 2000 Oct;7(19):1631-9. doi: 10.1038/sj.gt.3301281.
We report the isolation of a cosmid clone containing the entire human COL7A1 gene in one piece. The ability of the genomic sequences within this clone to direct tissue-specific expression of human collagen VII in transgenic mice was tested. The data show that the gene construct is capable of directing expression of collagen VII in the skin of fetal and neonatal transgenic mice. Expression of COL7A1 in these mice was widespread, in a pattern consistent with that found in human tissues and was in parallel with that of the endogenous mouse gene. Immunostaining, using human-specific antibodies, showed that human collagen VII protein was present at the skin basement membrane zone of the transgenic mice. Dermal extracts from 19-month-old transgenic mice contained mature human collagen VII protein, and fibroblasts derived from skin biopsies of these mice actively synthesized human collagen VII. The demonstration of successful and stable expression of human collagen VII in in vivo gene transfer is the first step towards the future development of therapeutic protocols for the rescue of keratinocyte function in severe blistering diseases such as dystrophic epidermolysis bullosa.
我们报告了一个粘粒克隆的分离，该克隆完整地包含了整个人类COL7A1基因。测试了该克隆内基因组序列在转基因小鼠中指导人类胶原蛋白VII组织特异性表达的能力。数据表明，该基因构建体能够在胎儿和新生转基因小鼠的皮肤中指导胶原蛋白VII的表达。这些小鼠中COL7A1的表达广泛，其模式与在人类组织中发现的一致，并且与内源性小鼠基因的表达平行。使用人特异性抗体进行免疫染色显示，人类胶原蛋白VII蛋白存在于转基因小鼠的皮肤基底膜区。19个月大的转基因小鼠的皮肤提取物中含有成熟的人类胶原蛋白VII蛋白，并且从这些小鼠的皮肤活检中获得的成纤维细胞能够积极合成人类胶原蛋白VII。在体内基因转移中成功且稳定地表达人类胶原蛋白VII的证明是朝着未来开发用于挽救严重水疱性疾病（如营养不良性大疱性表皮松解症）中角质形成细胞功能的治疗方案迈出的第一步。
