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crc press - E-Lib FK UWKS

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370 Cell-Penetrating Peptides: Processes and Applications<br />

An alternative method to electroporation was first described by Schutze–<br />

Redelmeier et al. 35 This group successfully transduced into T cells an antigenic<br />

peptide epitope subsequently presented at the cell surface in the MHC class I<br />

complex. Fenton et al. have also demonstrated the ability to transduce fluorescentlabeled<br />

Antp peptides into both quiescent and activated lymphocytes. 36 Our laboratory<br />

has expanded on this concept by transducing the Tat–E1A fusion protein<br />

(60 kDa) into ~100% of cells, both quiescent and cycling primary human lymphocytes<br />

in the population, in a concentration-dependent manner. The ability of Tat–E1A<br />

to bind endogenous p130 and pRB proteins (cellular targets of E1A) demonstrates<br />

that it retains its normal biological function. Moreover, this experiment was performed<br />

with a 2-year-old frozen preparation of Tat–E1A protein.<br />

Transduction has also been effective in determining the regulatory role of the<br />

small GTPase Rho in mediating integrin signaling events in avian osteoclasts. Small<br />

GTPases, such as CDC42, Rac, and Rho, regulate the cytoskeletal architecture of<br />

the cell, depending on the type of extracellular signals received. 37-39 For instance,<br />

Rho plays a dual role in formation of actin stress fibers and assembly of focal<br />

adhesions in fibroblasts. 38 Osteoclasts are important for bone remodeling processes<br />

and therefore are highly mobile cells. They achieve this state by rapidly changing<br />

their actin cytoskeletal attachment structures (called podosomes). 40,41 In vitro, these<br />

processes are stimulated by the growth factor osteopontin (OP). 42 Dissecting the role<br />

of Rho in podosome formation has been hampered by the inability to manipulate<br />

osteoclasts since these cells are essentially resistant to introduction of ex<strong>press</strong>ion<br />

constructs by transfection or retroviral infection.<br />

However, the use of Tat-mediated transduction has allowed these barriers to be<br />

bypassed. Constitutively active and dominant-negative forms of Tat–Rho protein<br />

were generated and added to osteoclast cultures that contained the protein, based<br />

on anti-hemagglutinin (HA) immunostaining and confocal microscopy. 43 The constitutively<br />

active Tat–Rho V14 rapidly (15 to 30 min) stimulated actin stress fiber<br />

formation in a manner indistinguishable from OP during the early phases of treatment.<br />

Consistent with this finding, the dominant-negative Tat–Rho blocked the<br />

podosome-forming effects of OP. Thus, the utility of Tat-mediated transduction<br />

allows for experimentation of primary cells, such as osteoclasts, that are extremely<br />

difficult to manipulate.<br />

Another powerful example of the utility of recombinant Tat fusion protein<br />

technology was the use of Tat–Cre recombinase to modify gene ex<strong>press</strong>ion in culture<br />

and to induce gene deletion in vivo (Becker and Dowdy, manuscript in preparation).<br />

Cre recombinase is a 38 kDa protein from bacteriophage P1 that mediates the sitespecific,<br />

intramolecular, or intermolecular recombination of DNA between a pair of<br />

13 bp inverted repeat sequences called loxP sites; thus it permits precise deletion or<br />

activation of genes. 44-46 For example, NIH 3T3 cells containing the actin promoter<br />

separated from the β-galactosidase transgene by a loxP-’stop’-loxP motif and thereby<br />

preventing its ex<strong>press</strong>ion were transduced with Tat–Cre protein. Unlike the poor<br />

efficiency afforded by DNA-based techniques to introduce Cre cDNA, transduction<br />

by Tat–Cre protein induced activation of β-galactosidase ex<strong>press</strong>ion and activity in<br />

more than 95% of cells within 24 h (Figure 17.1).

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