Embryonic Stem Cells as a Module for Systems Biology - Systems Biology: Networks, Models, and Applications - page 305

transfected relatively easily with high efficiency provides a means to
study gene responses and phenotypic changes in a high-throughput
micro-well platform. Such a maneuver is technically challenging or not
possible with other stem cells.
The advances made in molecular techniques that provide the ability
to control endogenous levels of protein and nucleic acids has revolu-
tionized our ability to study the over- or underexpression of a specific
gene. These include improvements in nonviral and viral-mediated cell
gene transfer vectors, expression modules, antisense oligonucleotides,
and RNA interference (RNAi) technologies [26-29]. Current methods
can achieve knockdown of RNA species that result in significant reduc-
tion of the proteins for which they code. The effect on ES cells can be
monitored by a combination of changes in morphology, cell death, pro-
liferation, or molecular markers indicative of differentiation. Profiling
of transcriptome changes can be global, performed with microarray
chips with high-density probes, or concentrated on monitoring embry-
onic lineage differentiation using focused chips or custom-designed
microfluidic cards (Applied Biosystems). Transgenic ES lines can also
be generated containing reporter genes, such as GFP (green fluorescent
protein) or luciferase, knocked into genes of interest (such as Oct4 ).
Several repositories with large collections of ES cell lines are now
available with heterozygous knockouts of single alleles using a random
genome-wide targeting with highly efficient promoter trap vectors
(Lexicon Genetics, http://www.lexicon-genetics.com; International
Genetrap Consortium, Sanger Center: http://www.igtc.org.uk/). An
investigator can browse these websites to order ES cell lines of interest
to study the loss-of-function mutation of the gene in the context of a
whole organism. Thus, altogether, there is an armamentarium of method-
ologies that are suitable for studying ES cell response that can be utilized
to study multiple gene interactions and test the potential of systems
biology approaches.
Expanding the Cellular Signaling Networks:
Transcription Mechanisms
Gene expression in eukaryotic cells is controlled by regulatory ele-
ments that recruit transcription factors with specific DNA recognition
properties. A major effort to advance understanding of gene regulation
is therefore directed at studies of transcription factors, their cognate
DNA binding sites, and protein-binding partners constituting coactivators
and suppressors of transcription. However, given the large number of
transcription factors, estimated at about 1500 in the mammalian genome,
we are only at the very elementary stage in identifying and characteriz-
ing the many components, and understanding the manner in which they
form interacting networks to modulate and coordinate gene expression.