Transient transfection of chick-embryo hepatocytes

Abstract 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 I footprinting and gel mobility-shift assays to identify the proteins that bind to those elements.