Biomedical Engineering Reference
In-Depth Information
two (or more) different samples. This allows an investigator not to deter-
mine which genes are expressed, but to compare the relative abundance
of gene expression in the different samples. This has proven critical to
following gene expression during development and differentiation of tis-
sues and cells, and has in the last few years been applied to analysis
of cancerous tissues. Importantly, these gene profiling studies have al-
lowed investigators to identify gene expression patterns that can distin-
guish between tumors that were once thought to be identical, and yet
responded differently to therapy. These profiles can not only be used to
predict how a person with a tumor will respond to therapy, but may ul-
timately provide new insights into the development of new therapeutics
for cancers refractory to treatment.
G. Promoter-protein interactions
Introduction
The human body is made up of over 200 types of cells, each of them
distinct. While they all contain the same genetic information, they each
developed as a result of the programming of a distinct subset of genes.
What makes a muscle a muscle and not a nerve is determined by the
pattern of gene expression prior to and during development, as well as
by the set of genes that are expressed in the differentiated cell. The
control of gene expression is a complex process that not only requires
that the promoter and enhancer elements that regulate expression are
“accessible” to transcription factors, but that the required transcription
factors are present. Determining which cis elements in a gene serve as
transcriptional control elements (the promoters and enhancers) requires
methods that can identify these sequences. Once identified, the identity
of the transcription factors that bind the sequences can be identified.
The following sections briefly outline some of the most common methods
employed.
Reporter assays
Reporter constructs are used to determine if a sequence of DNA
can act as a promoter or enhancer in a particular cell. The way that
reporter plasmids are constructed has been outlined in an earlier sec-
tion. These can be transiently transfected into the cell of interest, and
promoter activity measured using one of several “readouts” described
earlier including the production of luciferase, or or CAT activity. It is
important to remember that even if a particular sequence upstream of a
gene shows promoter activity in this type of assay it does not necessarily
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