Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs

Abstract

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.

Figure 1.

Construction of E. coli strain MW001. A β-lactamase (bla) recombinering cassette, containing a bla gene (green directional box) and homology arms (blue and purple boxes), corresponding to sequences flanking the tetA(C) gene (orange directional box) sequence in E. coli strain DY380, was PCR-amplified from a pFastBac1 restriction fragment template with primers 6030 and 6031 and used as donor DNA to replace, via recombineering, the tetA(C) gene in DY380 generating strain MW001.

Figure 2.

Generation of ‘long’ homology arm negative-selection recombineering replacement cassettes via In-Fusion cloning. (A) PCR products, generated from template bMON14272 and primer pairs 6054–6055 and 6059–6060, containing 3′ and 5′ gp64 flanking sequences were fused in an Overlap-PCR with primer pair 6054–6060 to produce a single PCR product with central, unique SnaBI site and terminal SmaI sites that was cloned into pGEM-T Easy to create pMW009. (B) RT-cassette and replacement cassettes, containing coding sequences for SeMNPV F, AdhoNPV F and AcMNPV gp64 genes, were generated by PCR, with respective primer pairs 6005a–6006a, 6009a–6010a, 6063–6064 and 6106–6107, and cloned, via In-Fusion cloning, into pMW009 generating, respectively, pMW012, pMW013, pMW033 and pMW024.

Figure 3.

Intramolecular recombination occurs between pairs of homologous regions in the baculoviral genome. (A) Electrophoretic separation of PCR products derived from amplification across regions predicted to be deleted in the 17 randomly selected clone DNAs, isolated following negative-selection recombineering of the RT-cassette-containing bMON14272-based clone bMW009 (lanes 1–17), using primer pairs 6190–6191 (lanes 1–9, Class I), 6184–6193 (lanes 10,11, Class II), 6183–6193 (lane 12, Class III), 6189–6191 (lanes 13–16, Class IV), 6188–6191 (lane 17, Class V). (B) Schematic representations of the deletion clone classes I–V illustrating (in the Class I deletion clone only) locations of homologous regions (hr) (red boxes), RT-cassette and mini-F ori (grey boxes), Kan^R gene (blue triangle). Annealing positions of primer pairs used to amplify across deleted regions and the remaining (grey segment) and deleted (white segment) segments in each clone class are also given.

Figure 4.

SeMNPV F- or AdhoNPV F-pseudotyped AcMNPV supports viable insect cell infection but are not able to transduce mammalian cells. (A) Essentially all cells of an Sf9 culture infected with CMV_PROM-p10_PROM::eGFP cassette-containing AcMNPV in which the native gp64 gene was replaced either with itself (vMW033), the SeMNPV F (vMW011) or the AdhoNPV F gene (vMW036) exhibited GFP expression indicating SeMNPV F- and AdhoNPV F-pseudotyped AcMNPV both mediate viable insect cell infection. All cultures were x3 passage. (B) In all cases <0.01% of cultured Saos-2 (Row 1), HepG2 (Row 2) or HeLa (Row 3) cells exhibit GFP expression 48 h following incubation with either vMW011 (50 pfu/cell) or vMW036 (100 pfu/cell). For illustration purposes fields of view containing rare GFP-expressing cells have been chosen. In contrast, >90% of these cells exhibit GFP expression following incubation with the control gp64 rescue virus vMW033 48 h post-incubation. Images were taken with a Nikon Eclipse TE2000-S inverted microscope with FITC filter set (100× magnification).