Biomedical Engineering Reference
In-Depth Information
any given time in a tissue. Some of the expressed genes will be “house-
keeping” genes, expressed in all tissues, and others may be responsible
for the development or function of a particular cell type. Together with
transcriptional profiling, differential screening is a powerful tool to un-
derstand lineage specific gene expression. However, identification of
a differentially expressed gene is only the first step. Once identified,
the important task is determining what the function(s) of these genes
might be the developmental fate or function of the cells. This is no small
task!
So how many genes are there? With the completion of the Human
Genome Project (and the completion of the sequencing of other mam-
malian genomes, including mouse and rat), one might have thought that
this would be a simple question. However, estimates of the number of
genes vary dramatically, from 24,000 to 75,000 genes, depending in
part on the algorithms used for the estimates. Most geneticists believe
the lower number is accurate and, if true, it means that humans only
have about 4,000 more genes than the simple nematode,
Caenorhab-
ditis elegans (C. elegans) a species that is frequently used as a model
system to study the role of genes in cell fate development.
There are a number of methods that have been developed over the
years to isolate and identify differentially expressed genes. Many of
these methods rely on screening cDNA libraries at some point. Each
of them have a number of drawbacks, and each comes with its own
set of artifacts. Nevertheless, these have proven to be powerful tools to
help scientists understand the role that differential gene expression has
in development and differentiation of cells and tissues.
Subtractive hybridization (also known as
subtractive cloning)
This was one of the first methods developed to identify differentially
expressed genes. This method is based on the ability of complementary
DNA (cDNA) to specifically hybridize to messenger RNA (mRNA). In its
simplest form, a large molar excess (10 to 100 fold) of mRNA from one
cell population (cell A) is hybridized to cDNA from another cell (cell B),
and the DNA-RNA hybrids removed. In theory, cDNAs that remain un-
hybridized represent transcripts of genes that are expressed in higher
abundance in cell type B. These can be used directly to screen a cDNA
library, or used as templates for generating double stranded cDNAs and
cloned. The resultant library represents differentially expressed genes.
Like all subtractive methods, subtractive hybridization is fraught with ar-
tifacts, and consequently the differential expression of any cloned gene
needs to be confirmed by a second method. A common method is to
Search WWH ::
Custom Search