Targeting expression with light using caged DNA
Targeting expression with light using caged DNA
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DOI：
10.1074/jbc.274.30.20895
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发表时间：
1999-07-23
影响因子：
4.8
通讯作者：
Haselton, FR
中科院分区：
文献类型：
--
作者：
Monroe, WT;McQuain, MM;Haselton, FR
在这份报告中，我们描述了使用光敏笼化合物的质粒表达的失活和位点特异性光诱导。将编码荧光素酶的质粒用1-（4，5-二甲氧基-2-硝基苯基）重氮乙烷（DMNPE）笼住，并用粒子轰击转染到大鼠皮肤的类似于1-cm直径的位点中。用笼状质粒转染的皮肤部位不表达荧光素酶，然而，随后将转染的皮肤部位暴露于355 nm激光诱导荧光素酶表达，与光量成比例。用DMNPE-笼状绿色荧光蛋白（GFP）质粒脂质体转染HeLa细胞显示类似的结果。用DMNPE笼化DNA在转录水平阻断表达，因为从线性化GFP质粒体外产生mRNA也被笼化阻断，随后通过暴露于光恢复。在这些实验的反应条件下，我们的吸光度数据表明，每个DMNPE笼状GFP质粒含有类似于270个笼状基团，除了抑制和随后恢复质粒生物活性外，这种相对少量笼状基团的存在和光切割也改变了质粒的电泳迁移率和光吸收特性。这种光诱导的表达策略提供了一种新的手段，靶向表达的遗传物质的空间和时间特异性。
In this report, we describe the inactivation and site-specific light induction of plasmid expression using a photosensitive caging compound. Plasmids coding for luciferase were caged with 1-(4,5- dimethoxy-2-nitrophenyl)diazoethane (DMNPE) and transfected into similar to 1-cm diameter sites of the skin of rats with particle bombardment. Skin sites transfected with caged plasmids did not express luciferase, However, subsequent exposure of transfected skin sites to 355-nm laser light induced luciferase expression in proportion to the amount of light. Liposome transfection of HeLa cells with DMNPE-caged green fluorescent protein (GFP) plasmids showed similar results. Caging DNA with DMNPE blocks expression at the level of transcription, since in vitro production of mRNA from linearized GFP plasmid was also blocked by caging and subsequently restored by exposure to light. Under the reaction conditions of these experiments, our absorbance data indicate that each DMNPE-caged GFP plasmid contains similar to 270 caging groups, In addition to inhibition and subsequent restoration of plasmid bioactivity, the presence and photocleavage of this relatively small number of cage groups also alters electrophoretic mobility of plasmids and optical absorption characteristics. This light-induced expression strategy provides a new means to target the expression of genetic material with spatial and temporal specificity.