to completing the fluorophore formation, takes tens of min-
utes, which possibly prevents a precise time-course analysis of
the target molecules. Another limitation is on the necessity to
introduce two expression plasmids of the two fluorescent pro-
tein fragments into the sample cells. The amount of reconsti-
tuted fluorescent protein generated in individual sample cells is
dependent on the expression levels of the respective fragments.
Controlling the expression of the respective two fragments in
individual cells is difficult when using a general technique on a
plasmid transfection method. This limitation might induce
misconceptions from quantitative analyses, especially of single
cells.- One method to avoid these limitations is to use a full-length
fluorescent protein with a very low expression level [10]. The
full-length fluorescent protein approach requires only a single
expression vector to generate the probe in target cells. As one
might expect, excess probe containing a full-length fluorescent
protein causes a severe background signal that critically ham-
pers the detection of target molecules. Therefore, the use of
full-length fluorescent protein-based probe requires stricter
control of the expression level of the probe. Consequently,
although an option to use full-length fluorescent protein
based probes is applicable, a split fluorescent protein reconsti-
tution approach is the prime choice for RNA probe design. - Based on these methods and principles, we designed TERRA
probe, in which GN and GC were fused respectively to the N-
terminal and C-terminus of mPUMt [12]. This probe alone
does not emit fluorescence. In the presence of TERRA, which
has a telomere repeat region, multiple probe molecules bind to
the repetitive region. Then, a GN portion and GC portions in
two adjacent probes cause a reconstitution reaction to yield
full-length EGFP. Consequently, the present probe fluores-
cently labels TERRA with low background. This strategy is
expected to be applicable to visualize various RNAs including
repetitive sequences. The following sections present our pro-
tocols of simultaneous observation of TERRA, telomeres, and
hnRNPA1 using the present TERRA probe (NLS-GN-
mPUMt-GC, Fig.2), a telomere probe (iRFP-TRF1), and
hnRNPA1-SNAPf.
3.2 Cell Sample
Preparation
3.2.1 Preparation of the
iRFP-TRF1 Expressing
U2OS Cell Line Through
Viral Infection
- Transfect the iRFP-TRF1/pCLNCX and the packaging vector
into Gag293 cells using Lipofectamine 2000™. - Change the cultivation medium to D-MEM containing 10%
FBS 8 h after transfection. - 24 h after transfection, take the supernatant containing viral
particles and mix it with 50% fresh medium and polybrene.
342 Hideaki Yoshimura and Takeaki Ozawa