TRANSIENT TRANSFECTION OF CHICK-EMBRYO HEPATOCYTES

Nutritional state regulates the expression of many genes via alteratio ns in the plasma levels of hormones or metabolic fuels. In many cases, transcription has been identified as the regulated step. The next obj ective in the experimental analysis of these transcriptionally regulat ed genes is to identify the cis-acting sequence elements that confer r egulation of a specific gene by a particular hormone or agent. One met hod for identifying cis-acting sequence elements involves transient ex pression of transgenes introduced into responsive cell types by a proc ess called transfection. Promoter/regulatory sequences from the gene o f interest are ligated to a reporter gene, and the chimeric DNA is add ed to cells in culture. Under appropriate conditions the DNA enters th e cell, migrates to the nucleus, and is transcribed, but is not integr ated into chromosomal DNA. Expression of the reporter gene is monitore d to assess function of the putative promoter/regulatory DNA. The repo rter gene codes for a protein that is not normally expressed in the re sponsive cell type. If this protein is an enzyme, then the amount of i ts activity is a measure of the ability of cis-acting elements in the promoter/regulatory DNA to regulate transcription. If a specific fragm ent of DNA can confer hormone responsiveness on the expression of the reporter gene, then sequences containing 5' or 3' deletions or mutatio ns 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 ass ay'' can then be used in DNase 1 footprinting and gel mobility-shift a ssays to identify the proteins that bind to those elements.