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Protein Transport 371
LacZ LoxP :
% Recombination
100
75
50
25
FIGURE 17.1 Recombination of LoxP sites by transducible Tat–Cre protein. NIH 3T3 harboring
a transgene comprised of a promoter separated from the β-galactosidase gene by a
LoxP-stop-LoxP motif was treated with Tat–Cre protein. Treated cells were assayed for
β-galactosidase 24 h post-treatment by staining with X-gal.
Tat–Cre has also been used on primary splenocytes harvested from retinoblastoma
loxP mice to cause recombination-specific excision of exon 19 from the
retinoblastoma gene. After overnight incubation, PCR analysis and subsequent
sequencing of the exon 19 region showed that, predominantly, all cells in culture
contained the specific deletion of DNA sequence from exon 19 surrounding the loxP
recombination sites, while cells treated with Cre were not recombined (Becker–
Hapak et al., unpublished results). Moreover, these results could be reproduced in
vivo following intraperitoneal administration of Tat–Cre and were only limited by
proteolytic degradation in the serum. In both examples, exogenously applied Tat–Cre
was able to pass through the plasma membrane, accumulate within the nucleus, and
catalyze site-specific recombination between the loxP sites.
17.4.2 IN VIVO APPLICATIONS
0
ctrl
LoxP LoxP
(a)
3T3-LacZ LoxP cells
(b)
TAT-CRE
LacZ
As with any potential pharmacological approach, one must translate in vitro findings
to animal models. Can PTDs deliver molecules into cells of entire organisms? Based
on past and recent studies in model organisms, the answer is a resounding yes. In
1994, Fawell et al. 47 demonstrated a limited, but significant, capacity of Tat fusions
to enter tissues in vivo in mice. Schutze–Redelmeier et al. 35 showed that intraperi-