TRANSIENT TRANSFECTION OF CHICK-EMBRYO HEPATOCYTES
TRANSIENT TRANSFECTION OF CHICK-EMBRYO HEPATOCYTES
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DOI：
10.1016/0955-2863(93)90074-7
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发表时间：
1993-07-01
影响因子：
5.6
通讯作者：
GOODRIDGE, AG
中科院分区：
文献类型：
--
作者：
BAILLIE, RA;KLAUTKY, SA;GOODRIDGE, AG
营养状态通过改变血浆中激素或代谢燃料水平来调节许多基因的表达。在许多情况下，转录已被确定为受调控的步骤。这些转录调节基因的实验分析的下一个目标是鉴定顺式作用序列元件，这些元件赋予特定激素或试剂对特定基因的调节。一种鉴定顺式作用序列元件的方法涉及通过称为转染的过程引入响应细胞类型的转基因的瞬时表达。将来自感兴趣基因的启动子/调节序列连接到报告基因，并将嵌合 DNA 添加到培养的细胞中。在适当的条件下，DNA 进入细胞，迁移到细胞核并进行转录，但不会整合到染色体 DNA 中。监测报告基因的表达以评估推定启动子/调节DNA的功能。报告基因编码的蛋白质通常在反应细胞类型中不表达。如果该蛋白质是酶，则其活性量是启动子/调节 DNA 中顺式作用元件调节转录能力的量度。如果特定的 DNA 片段可以赋予报告基因表达激素反应性，则使用可疑调控元件中包含 5' 或 3' 缺失或突变的序列来更具体地鉴定序列元件。这些 DNA 序列是调节蛋白的结合位点。然后，在此“功能测定”中鉴定的序列可用于 DNase 1 足迹和凝胶迁移率测定，以鉴定与这些元件结合的蛋白质。
Nutritional state regulates the expression of many genes via alterations in the plasma levels of hormones or metabolic fuels. In many cases, transcription has been identified as the regulated step. The next objective in the experimental analysis of these transcriptionally regulated genes is to identify the cis-acting sequence elements that confer regulation of a specific gene by a particular hormone or agent. One method for identifying cis-acting sequence elements involves transient expression of transgenes introduced into responsive cell types by a process called transfection. Promoter/regulatory sequences from the gene of interest are ligated to a reporter gene, and the chimeric DNA is added to cells in culture. Under appropriate conditions the DNA enters the cell, migrates to the nucleus, and is transcribed, but is not integrated into chromosomal DNA. Expression of the reporter gene is monitored to assess function of the putative promoter/regulatory DNA. The reporter gene codes for a protein that is not normally expressed in the responsive cell type. If this protein is an enzyme, then the amount of its activity is a measure of the ability of cis-acting elements in the promoter/regulatory DNA to regulate transcription. If a specific fragment of DNA can confer hormone responsiveness on the expression of the reporter gene, then sequences containing 5' or 3' deletions or mutations in suspected regulatory elements are used to identify the sequence elements more specifically. These DNA sequences are binding sites for regulatory proteins. The sequences identified in this ''functional assay'' can then be used in DNase 1 footprinting and gel mobility-shift assays to identify the proteins that bind to those elements.