Biomedical Engineering Reference
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approach for the detection of RNAs coupled to gold particles in vivo and has the
advantage of visualising cellular components at the ultra-structural level. Refinement
of the technique has seen the use of antisense probes to specific RNAs, labelled with
antibodies conjugated to gold. Different size gold particles allow several RNAs and/
or proteins to be visualised simultaneously (Herpers et al.
2010
; Delanoue et al.
2007
; Herpers and Rabouille
2004
). The visualisation of mRNA in fixed material
continues to be an essential technique for investigating the localisation of mRNAs
and colocalisation with protein factors and cell structures. However, in order to
reveal the underlying mechanisms underlying RNA localisation and to observe the
RNA on its journey rather than just at its destination the RNA must be visualised in
real time using live cell imaging techniques.
For large cells, often the simplest method for visualising mRNA with live cell
imaging is via the micro-injection of fluorescently labelled RNA. The RNA is syn-
thesised in vitro and can be conjugated to a fluorophore of choice. Careful selection
of fluorophores can allow several RNAs to be visualised simultaneously through
multi-colour imaging. This technique has been used to image
Vg1
mRNA localisa-
tion in the
Xenopus
oocyte (Yisraeli et al.
1990
) ,
oskar
localisation in the
Drosophila
oocyte and to show that
wingless
and the
pair-rule
transcripts localise along micro-
tubules and require the molecular motor Dynein (Wilkie and Davis
2001
) . Micro-
injection has also been used to map
cis
-acting signals in
gurken
and
I
factor mRNAs
in
Drosophila
oocytes and embryos (Van De Bor et al.
2005
) and identify protein
transacting factors, e.g. Egalitarian, required for RNA localisation (Bullock and Ish-
Horowicz
2001
). The main disadvantage of introducing in vitro transcribed
fluorescently labelled RNA into a cell is that the endogenous RNA is not labelled.
The MS2 system for visualising native mRNAs in vivo was originally developed to
label
Ash1
mRNA in
S. cerevisiae
(Bertrand et al.
1998
) . mRNAs are genetically
engineered to contain multiple copies of the MS2 loop, which then bind an MS2-
coat protein fused to green fluorescent protein (MCP-GFP), also genetically engi-
neered to be expressed. The MS2-coat protein specifically binds the MS2 loops in
the RNA with high affinity. Typically 6, 12 or 18 copies of the MS2 loops in the
RNA ensure a bright fluorescent signal from each RNA copy for live cell imaging.
The MS2 system has been successfully employed for the visualisation of
nanos
,
gurken
,
bicoid
and
oskar
mRNAs in
Drosophila
(Forrest and Gavis
2003
; Jaramillo
et al.
2008
; Weil et al.
2006
; Zimyanin et al.
2008
) .
Molecular motor-based RNA localisation is reliant on the cells underlying
cytoskeleton. As well as localisation occurring via the microtubule network, actin
filaments are also of importance. For example actin mRNA is transported on the
actin network, and
oskar
mRNA anchoring is actin dependent (St Johnston
2005
) .
In the
Drosophila
oocyte, the microtubule network can be visualised with the EB1
protein labelling the growing plus end of microtubules. By imaging and tracking
these microtubule tips and the RNA cargoes it has been possible to unravel the
mechanisms of cell polarisation (Zimyanin et al.
2008
) . Visualisation of the micro-
tubule network appears random, highly dynamic and undirected. However, through
the use of particle tracking and analysis tools the polarity of the microtubule net-
work can be unravelled (Parton et al.
2011
; Hamilton et al.
2010
) .
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