Biology Reference
In-Depth Information
Fig. 1
Schematic outline of the homologous recombination in
E. coli
(procedure I). The shuttle plasmid already
contains the gene of interest, which is fl anked by the 5
) in one end, and adeno-
viral sequences in the other. The pKP1.4 backbone plasmid contains the adenovirus genome (except the E1
region). First, the pKP1.4 plasmid is linearized by
Swa
I, and the shuttle plasmid is digested by one (RE-I) or two
restriction enzymes (RE-II) in the Amp
R
gene. Both digested plasmids are co-transfected in BJ5183 bacteria for
homologous recombination and only bacteria carrying recombinant plasmids containing the adenoviral
genome plus the gene of interest are viable in LB + Amp
R
plates. For production of the viral pre-stock, recom-
binant plasmids are digested with
Pac
I to liberate the vector genome
′
ITR and packaging signal (
Ψ
