Biomedical Engineering Reference
In-Depth Information
Probes
To localize a particular protein or gene of interest, a probe containing either
a homologous sequence or primary/secondary antibody for the gene is
developed. In addition to this target recognition domain, probes also con-
tain a label or dye to allow for easy location.
Fluorescent dyes may be used to label proteins and genetic material
itself. Fluorescent dyes such as Fluorescein, Rhodamine, and Texas Red are
capable of binding to DNA
in situ
. A comprehensive review provides an
excellent guide to choosing the proper fl uorescent protein (Haugland, 1992;
Shaner
et al.
, 2005).
Vectors are currently available from several vendors (MD Biosciences
Clontech, Qbiogene, Stratagene, Clontech, Vector Labs) with GFP-coding
regions. The fi rst major consideration is where the GFP tag should be
placed, as it is a relatively large protein at 27 kD. Often it is advisable to
begin with several attachment sites to determine empirically where the best
results are obtained. The promoters supplied with commercially available
vectors are often highly expressive, such as cytomegalovirus (CMV) and
may not be universally applicable. For these cases, weaker promoters in
addition to other modifi cations to probe design can be used to avoid over-
saturation of the sample (Tsukamoto
et al.
, 2000). Other promoters can be
used for inducible expression as well (Rubinchik
et al.
, 2001; Valdivia and
Falkow, 1996). Linker sequences can also be utilized to help isolate the func-
tional GFP portion of the protein. Linkers can be designed in a number of
ways with different sizes and cleavage sites (Spector and Goldman, 2006;
Wahlfors
et al.
, 2001).
Targeted gene expression
Depending on the mode and gene of interest, gene expression can vary in
intensity and duration. Different modes of transduction are recommended
for each application (Table 4.9). It is also important to note that the expres-
sion of episomal genes will often not be representative of physiological
levels.
Permanent integration of a probe sequence can be achieved through incor-
poration into host genome. Each new application requires careful optimization
steps including probe concentration, promoter strength, and timeline of analy-
sis. Protocols for the development of a stable mammalian cell line expressing
GFP fusion proteins have been developed (Strukov and Belmont, 2005).
Following the development of a stable cell line, it is critical to correctly iden-
tify the localization of the protein of interest in the clonal population. Adequate
localization can be determined via fl uorescence microscopy. If localization has
