Skip to main content
. 2022 Nov 16;13(11):2129. doi: 10.3390/genes13112129

Figure 2.

Figure 2

Homologous recombination strategy for adenovirus genome engineering. (A). The three types of selection marker (SM)-containing PCR templates for counter-selection are displayed. (1) SM only; (2) SM with a single flanking I-PpoI site; (3) SM with I-PpoI flanking both ends of the SM, for detail, see also Figure S1. (B). The three counter-selection strategies with different SM PCR-templates. All strategies shared the same first step: ampicillin-mediated positive selection to insert the selection marker cassette (ccdB-Amp) generated from different PCR-templates as shown in A. The small pink rectangles next to SM in B (2) and (3) show the I-PpoI sites. The adenoviral genome containing plasmid and the ccdB-Amp cassette flanked by homologous arms to the target region of the adenoviral genome are co-electroporated into the E. coli strain GBRed-GyrA462, in which the λ Red–mediated linear–circular homologous recombination (LCHR) takes place to insert the ccdB-Amp cassette into the desired site. The three strategies differ on the counter-selection step: (1) counter-selection takes place in E. coli strain GBRed, the adenoviral genome containing plasmid with previously inserted ccdB-Amp cassette, and the insert flanked by homologous arm to target region of adenoviral genome are co-electroporated. Then, the λ Red-mediated linear-circular homologous recombination (LCHR) replaces the ccdB-Amp cassette with insert. In contrast to strategy (1), the counter-selection marker containing adenovirus plasmids are pre-cut with I-PpoI in the strategies (2) and (3), which expose the homologous arm on one (2) or both sites (3). Then, the linearized adenovirus genome containing plasmid, and the insert flanked by homologous arms to the target region of the adenoviral genome are co-electroporated into the E. coli strain GB05-dir, in which the RecET recombinase is induced with L-arabinose to express, enabling linear-linear homologous recombination (LLHR) to replaces the ccdB-Amp cassette with aimed insert. p15A, p15A plasmid origin of replication [13]; chl, chloramphenicol acetyltransferase, chloramphenicol resistant gene; SM, ccdB- ampicillin double selection marker; pR6K, plasmid origin of replication that requires pi gene for replication [17,18]; HAF/R, homologous arm forward/reverse; gAd, adenovirus genome. Figures are created with BioRender.com.