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370 Cell-Penetrating Peptides: Processes and Applications
An alternative method to electroporation was first described by Schutze–
Redelmeier et al. 35 This group successfully transduced into T cells an antigenic
peptide epitope subsequently presented at the cell surface in the MHC class I
complex. Fenton et al. have also demonstrated the ability to transduce fluorescentlabeled
Antp peptides into both quiescent and activated lymphocytes. 36 Our laboratory
has expanded on this concept by transducing the Tat–E1A fusion protein
(60 kDa) into ~100% of cells, both quiescent and cycling primary human lymphocytes
in the population, in a concentration-dependent manner. The ability of Tat–E1A
to bind endogenous p130 and pRB proteins (cellular targets of E1A) demonstrates
that it retains its normal biological function. Moreover, this experiment was performed
with a 2-year-old frozen preparation of Tat–E1A protein.
Transduction has also been effective in determining the regulatory role of the
small GTPase Rho in mediating integrin signaling events in avian osteoclasts. Small
GTPases, such as CDC42, Rac, and Rho, regulate the cytoskeletal architecture of
the cell, depending on the type of extracellular signals received. 37-39 For instance,
Rho plays a dual role in formation of actin stress fibers and assembly of focal
adhesions in fibroblasts. 38 Osteoclasts are important for bone remodeling processes
and therefore are highly mobile cells. They achieve this state by rapidly changing
their actin cytoskeletal attachment structures (called podosomes). 40,41 In vitro, these
processes are stimulated by the growth factor osteopontin (OP). 42 Dissecting the role
of Rho in podosome formation has been hampered by the inability to manipulate
osteoclasts since these cells are essentially resistant to introduction of expression
constructs by transfection or retroviral infection.
However, the use of Tat-mediated transduction has allowed these barriers to be
bypassed. Constitutively active and dominant-negative forms of Tat–Rho protein
were generated and added to osteoclast cultures that contained the protein, based
on anti-hemagglutinin (HA) immunostaining and confocal microscopy. 43 The constitutively
active Tat–Rho V14 rapidly (15 to 30 min) stimulated actin stress fiber
formation in a manner indistinguishable from OP during the early phases of treatment.
Consistent with this finding, the dominant-negative Tat–Rho blocked the
podosome-forming effects of OP. Thus, the utility of Tat-mediated transduction
allows for experimentation of primary cells, such as osteoclasts, that are extremely
difficult to manipulate.
Another powerful example of the utility of recombinant Tat fusion protein
technology was the use of Tat–Cre recombinase to modify gene expression in culture
and to induce gene deletion in vivo (Becker and Dowdy, manuscript in preparation).
Cre recombinase is a 38 kDa protein from bacteriophage P1 that mediates the sitespecific,
intramolecular, or intermolecular recombination of DNA between a pair of
13 bp inverted repeat sequences called loxP sites; thus it permits precise deletion or
activation of genes. 44-46 For example, NIH 3T3 cells containing the actin promoter
separated from the β-galactosidase transgene by a loxP-’stop’-loxP motif and thereby
preventing its expression were transduced with Tat–Cre protein. Unlike the poor
efficiency afforded by DNA-based techniques to introduce Cre cDNA, transduction
by Tat–Cre protein induced activation of β-galactosidase expression and activity in
more than 95% of cells within 24 h (Figure 17.1).