Advances in transgenic technology - Principles of Gene Manipulation - page 255

Box 13.1 continued

1999, Ikawa et al. 1999, Naylor 1999). GFP is a
bioluminescent marker that causes cells to emit
bright green fluorescence when exposed to blue or
ultraviolet light. However, unlike luciferase, GFP has
no substrate requirements and can therefore be used
as a vital marker to assay cellular processes in real
time. Other advantages of the molecule include the
fact that it is non-toxic, it does not interfere with
normal cellular activity and it is stable even under
harsh conditions (Ward & Bokman 1982).
GFP was first used as a heterologous marker
in Caenorhabditis elegans (Chalfie et al. 1994).
However, early experiments with GFP expression in
a variety of other organisms, including Drosophila
(Wang & Hazelrigg 1994), mammalian cell lines
(Marshall et al. 1995) and plants (Haseloff &
Amos 1995, Hu & Chen 1995, Sheen et al. 1995),
identified a number of difficulties in the heterologous
expression of the gfp gene. Modifications have been
necessary for robust GFP expression in some plants
(Chiu et al. 1996). In Arabidopsis , for example, the
original gfp gene is expressed very poorly due to
aberrant splicing. This problem was addressed by
removing a cryptic splice site recognized in this plant
(Haseloff et al. 1997). The original gfp gene has
been extensively modified to alter various properties
of the protein, such as the excitation and emission
wavelengths, to increase the signal strength and to
reduce photobleaching (e.g. Heim & Tsein 1996,

Zolotukhin et al. 1996, Cormack et al. 1997). As a
result, a range of variant GFPs are available which
can be used for dual labelling (e.g. Tsien & Miyawaki
1998; reviewed by Ellenberg et al. 1999). Fluorescent
proteins of other colours are also available, including
a red fluorescent protein from Anthozoa (Matz et al.
1999). A mutant form of this protein changes from
green to red fluorescence over time, allowing it to
be used to characterize temporal gene expression
patterns (Terskikh et al. 2000).
GFP is particularly useful for generating fusion
proteins, providing a tag to localize recombinant
proteins in the cell. This facilitates the investigation
of intracellular protein trafficking, and even the
transport of proteins between cells. An early example
of this application was the use of GFP to monitor the
movement of ribonucleprotein particles during
oogenesis in Drosophila (Wang & Hazelrigg 1994).
Kohler et al. (1997) have used GFP to study the
exchange of molecules between plant organelles,
while Wacker et al. (1997) have investigated the
transport of a GFP-tagged protein along the secretory
pathway. The use of GFP to study the real-time
dynamics of a systemic viral infection in plants
was described by Padgett et al. (1996).

* Abbreviations: ONPG O-nitrophenyl- β -d-galactopyranoside;
MUG 4-methylumbelliferyl- β -d-galactoside; X-gal:
5-bromo-4-chloro-3-indolyl- β -d-galactopyranoside; X-gluc:
5-bromo-4-chloro-3-indolyl- β -d-glucuronic acid.

recombination in several important respects. In
terms of gene manipulation, the most important
differences between these processes concern the
availability of the recombinase and the size and
specificity of its target sequence. Homologous re-
combination is a ubiquitous process that relies on
endogenous recombinase enzymes present in every
cell, whereas site-specific recombination systems are
very specialized and different systems are found
in different organisms. Homologous recombination
occurs between DNA sequences with long regions of
homology but no particular sequence specificity,
whereas site-specific recombination occurs at short,
specific recognition sites. This means that target

sites for site-specific recombination can be intro-
duced easily and unobtrusively into transgenes, but
recombination will only occur in a heterologous cell
if a source of recombinase is also supplied. The power
of site-specific recombination as a tool for genome
manipulation thus relies on the ability of the experi-
menter to supply the recombinase enzyme on a con-
ditional basis.
A number of different site-specific recombination
systems have been identified and several have been
studied in detail (reviewed by Craig 1988, Sadowski
1993). Some recombinases, such as bacteriophage
λ integrase, require various accessory proteins for

efficient recombination. However, the simplest systems