Biomedical Engineering Reference
In-Depth Information
Since the discovery of the GFP in 1961 by Osamu Shimomura and
colleagues [7], 30 years had to pass before the gene for GFP was cloned and
the 238 amino acids sequence determined by Prasher [8]. Douglas Prasher was
the first person to realize the potential of GFP as a biomarker for proteins in
cells [9]. The manufacture of proteins using the instructions from the gene is
called protein expression. Prasher envisioned that it would be possible to use
biomolecular techniques to insert the GFP gene at the end of the gene of any
protein, right before the stop codon (Fig. 10.2, left). Encoded in the DNA is
some type of index that directs the molecular machinery to the start of the
gene of each necessary protein. When new protein is required, the gene is read,
and the protein is manufactured. At the end of the gene is a message called a
stop codon, which ends protein production. When the cell needs to make that
protein, it would go to the specific gene, use the information encoded in the
gene to make it, but instead of stopping when the protein was made, this cell
would carry on making GFP until it reached the stop codon at the end of the
GFP gene. As a result, the cell would produce a “chimeric” (e.g., modified)
protein with a GFP attached to it.
A variety of techniques have been developed to construct FP fusion prod-
ucts and enhance their expression in mammalian and other systems. The
primary vehicles for introducing FP chimeric gene sequences into cells are
genetically-engineered bacterial plasmids and virus that act as vectors and
transfer the genetic information into the cell either transiently or stably
(Fig. 10.2, right). In transient, or temporary, gene transfer experiments (of-
ten referred to as transient transfection), plasmid or viral DNA introduced
into the host organism does not necessarily integrate into the chromosomes
of the host cell, but can be expressed in the cytoplasm for a short period of
plasmid
vector
DNA
endocytosis
endosome
DNA-lipid
complex
released
DNA
gene
for protein
gene
for protein
lipofection
reagent
cell
membrane
gene for GFP
stop code
for protein
stop code
for protein
Fig. 10.2.
Left
: Fusion Construct. Adapted from [10].
Right
: Lipid-mediated trans-
fection in mammalian cells
