Biology Reference
In-Depth Information
Fig. 1
(a)
Schematic representation of the modifi ed recombineering: The 1st targeting delivers a galK-Kn cas-
sette (combined
gray
and
black boxes
) over red mediated recombination in SW102 strain to targeted BAC
sequence (
hatched box
) between the selected homologies (indicated by
white boxes
and H1 and H2). For this,
a PCR-derived linear fragment carrying the galK-Kn cassette fl anked by the same homology regions is used.
BAC vector is indicated by a
dotted box
. The resulting intermediate can be selected by kanamycin selection
(Kn). The second targeting delivers a double-stranded linear DNA fragment carrying a sequence with desired
mutation (
diamond patterned box
) fl anked by the same homology regions (H1 and H2). This will replace the
gakK-Kn cassette thereby rendering the mutant BAC Gal-, which can survive the 2-deoxy-galactose (DOG)
counter selection, as it suppresses the growth of the Gal+ clones carrying the intermediate constructs.
(
b
) Design of homology-fl anked primers for amplifi cation of
galK-kan
cassette: the sequence to be modifi ed
(
hatched box
, wt) is fl anked by up and downstream homologies (H1 and H2, respectively). For the fi rst targeting
(1st) pgalK-Kn cassette is amplifi ed by homology-fl anked primers, which consist of two parts. In the upper
primer, 50 nucleotide from the upper homology strand H1 (fh) is fused to the priming sequence, which is spe-
cifi c to the 5
end of the galK-Kn cassette (fp). In the lower primer, 50 nucleotide from the complementary
homology strand H2 (rh) is fused to the 5
′
end of the galK-
Kn cassette (rp). In the same way, PCR fragment for the second targeting (2nd) is amplifi ed by homology-
fl anked primers, which contains the same parts. In the upper primer, 50 nucleotide from the upper homology
strand H1 (fh) is fused to the priming sequence, specifi c to the 5
′
of the priming sequence, which is specifi c to the 3
′
end of the mutant sequence (diamond pat-
terned box) (sp). In the lower primer, 50 nucleotide from the complementary homology strand H2 (rh) is fused
to the 5
′
end of the mutant sequence (rs). (
c
) Map of the
marker template plasmid pgalK-Kn. Priming sites (used for the design of the homology-fl anked primers in the
fi rst targeting) are indicated by
arrows
(fp and rp). (
d
) Schematic representation of the recombineering target-
ing intermediate BAC construct pB-TA5, which can be used to construct Ad5-BAC from genomic Ad DNA. Here
the Ad left and right ITRs (L-IRT and R-ITR) serve as homologies for the replacement of the galK-Kn cassette
by the genomic Ad DNA
′
of the priming sequence, which is specifi c to the 3
′
