7.3.1.2. CreatorTMCloning.The Creator cloning system is an alternative approach that
allows the efficient transfer of DNA fragments from donor vectors to “creator” expression
vectors. This transfer is mediated by the Cre-loxPsite-specific recombination reaction dis-
covered in bacteriophage P1 (Sternberg and Hamilton 1981). First, PCR products are
inserted into Creator-compatible donor vectors using a proprietary enzyme called In-
FusionTM, creating “master” clones (Fig. 7.14).
LoxPsites flanking the insertion site of the master clone allow cloned DNA fragments to
be transferred to a singleloxPsite in an acceptor vector. This second transfer relies on the
presence of Cre recombinase. TheloxPsites contain inverted repeat sequences separated by
a spacer region, within which DNA breakage and reunion take place. To select the correct
recombination product, as with the Gateway system, both positive and negative selection is
used. Donor vectors, or recombinants that retain the donor vector backbone, are selected
against because they contain asacBgene fromBacillus subtilisthat generates a toxic metab-
olite in the presence of sucrose— recombinants containing the CAT gene are selected for by
the presence of chloramphenicol in the medium. Using this dual-selection regime, only bac-
teria containing the desired recombinant construct that lacks thesacBgene and contains the
appropriate antibiotic resistance marker gene will survive in the presence of sucrose
(Fig. 7.15).
7.3.1.3. Univector (EchoTM) Cloning.The Univector system was developed by Liu
and coworkers (Liu et al. 1998, 2000). Like the clonetech CreatorTMsystem, the Univector
Figure 7.14.The PCR products to be cloned are generated with gene- or promoter-specific primers
that contain 15-bp extensions. In this hypothetical example a cDNA is amplified. These extensions are
homologous to a region of the vector that flanks any unique restriction site. The In-FusionTMenzyme
creates single-stranded regions that share homology between the vector and the PCR product, allowing
the PCR product to join the specialized “donor” vector by strand displacement and In-Fusion-
mediated recombination. In this hypothetical example, the cDNA is inserted upstream of a plant ter-
minator sequence in the “donor” vector.
7.3. GREATER DEMANDS LEAD TO INNOVATION 175