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Signal Sequence-Based Cell-Penetrating Peptides 107
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24h 12h Baf-A Chl Cy-B 4∞C
(a) (b)
FIGURE 5.4 (Color Figure 5.4 follows p. 14.) MPG-mediated gene delivery mechanism.
(a) MPG-mediated pcDNA3.1NT-GFP plasmid delivery in human fibroblast cell line HS-68.
Cells were incubated in the presence of preformed MPG/DNA complexes for 1 h at 37°C,
after which they were replaced in DMEM supplemented with 10% FCS. Expression of GFP
is monitored 24 h later. (b) Mechanism of MPG-mediated gene delivery. Cells were incubated
in the presence of preformed MPG/DNA (in red) complexes for 1 h at 4°C, or at 37°C in the
absence or presence of several cell-trafficking inhibitors, bafilomycin A (Baf-A), chloroquin
(Chl), and cytochalasin B (Cy-B). Following this treatment, cells were replaced in DMEM
supplemented with 10% FCS and expression of GFP was monitored 12 or 24 h later. As a
control, similar experiments were performed with a cationic lipid formulation (in blue).
noncovalently attached to plasmid DNA was shown to improve mammalian cell
transfection significantly. Both NLS and electrostatic properties can be used to
improve the effect of the NLS. M9 sequence, fused to a scrambled NLS for DNA
binding and combined with Lipofectamine TM for transfection increases reporter gene
expression ten-fold compared to scrambled M9 sequence. 85 Associating an NLS with
a PNA is an excellent transfection tool, and NLS–PNA conjugates have been shown
to be specific using scrambled NLS as a negative control. 66
Several methods have been described to study the nuclear transfer of cellpenetrating
peptide containing NLS.
1. Interaction with importin factor. Physical interaction with import factors
can be monitored in vitro using recombinant proteins which yield information
concerning specificity, affinity, and stoechiometry. Methods have
been described using recombinant importin proteins and an ELISA-based
binding assay. 33 In vitro binding assays reporting the binding of NLS to
importin have revealed that the NLS is specific.
2. Microinjection and cell permeabilization. The direct effect of an NLS on
nuclear transfer can be investigated by either microinjection into the
cytoplasm or using methods for cell permeabilization. Digitonin permeabilization
of the cell and direct injection of genetic material have been
the methods of choice for study of NLS function. 71,94 However, these are
limited by the quality and quantity of the preparation. DNA–NLS complexes