Nucleofection: a new method for cutaneous gene transfer?
Nucleofection: a new method for cutaneous gene transfer?
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DOI：
10.1155/jbb/2006/26060
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发表时间：
2006
影响因子：
--
通讯作者：
Steinstraesser L
中科院分区：
文献类型：
--
作者：
Jacobsen F;Mertens-Rill J;Beller J;Hirsch T;Daigeler A;Langer S;Lehnhardt M;Steinau HU;Steinstraesser L
背景在原代上皮细胞中非病毒基因转移后的转染效率是有限的。本研究的目的是比较最近可用的核转染方法与已建立的转染试剂FuGENE 6的转染效率。方法.通过FuGENE 6或Amaxa Nucleofector装置用报告基因构建体转染原代人角质形成细胞（HKC）、原代人成纤维细胞（HFB）和人角质形成细胞系（HaCaT）。在相应的时间点，测定β-半乳糖苷酶表达、细胞增殖（MTT-试验）、转导效率（X-gal染色）、细胞形态和细胞毒性（CASY）。 结果核转染后的转基因表达在HKC和HFB中显着更高，并且比FuGENE 6更早检测到（3小时对24小时）。脂质体转染后，80%-90%的细胞保持增殖，而对细胞形态没有任何影响。相比之下，核转染导致角质形成细胞大小减少，仅20%-42%增殖细胞。 结论与细胞毒性的方法依赖性增加相关，核转染后的转基因表达比脂质转染后更早且更高。
Background. Transfection efficacy after nonviral gene transfer in primary epithelial cells is limited. The aim of this study was to compare transfection efficacy of the recently available method of nucleofection with the established transfection reagent FuGENE6. Methods. Primary human keratinocytes (HKC), primary human fibroblasts (HFB), and a human keratinocyte cell line (HaCaT) were transfected with reporter gene construct by FuGENE6 or Amaxa Nucleofector device. At corresponding time points, β-galactosidase expression, cell proliferation (MTT-Test), transduction efficiency (X-gal staining), cell morphology, and cytotoxicity (CASY) were determined. Results. Transgene expression after nucleofection was significantly higher in HKC and HFB and detected earlier (3 h vs. 24 h) than in FuGENE6. After lipofection 80%–90% of the cells remained proliferative without any influence on cell morphology. In contrast, nucleofection led to a decrease in keratinocyte cell size, with only 20%–42% proliferative cells. Conclusion. Related to the method-dependent increase of cytotoxicity, transgene expression after nucleofection was earlier and higher than after lipofection.