Red/ET recombination was performed as follows: a zeocin resistance cassette and an FRT-flanked kanamycin/neomycin resistance cassette were PCR-amplifiedwith primers PCR1–5′ and PCR1–3′ or PCR2–5′ and PCR2–3′ (Supplementary Table 1). With a goal of generating a BAC transgene harboring truncated N171 98Q htt cDNA, the FRT-flanked neomycin resistance cassette was also amplified with primers PCR3–5′ and PCR3–3′, which contained homology arms mediating integration into full-length 98Q htt cDNA downstream of codon 246, thereby deleting the remaining htt cDNA. In a first Red/ET recombination step, 200 ng of PCR-product 1 were electroporated into E. coli cells harboring pRed/ET and pFL98Q to generate pFL98Q1. ≥200 ampicillin- and zeocin-resistant colonies were obtained from the electroporated cultures that had been induced with L-arabinose, while no colonies were obtained from the non-induced controls. Individual clones were propagated and plasmid DNA was isolated and analyzed by restriction digest. Basepair-precise modification of pFL98Q was confirmed by sequencing. In a second Red/ET recombination step, 200 ng of PCR-products 2 or 3 were electroporated into E. coli cells harboring pFL98Q1 and pRed/ET to generate pFL98Q2 and pFL98Q2-stop, respectively. As above, ≥200 ampicillin-, zeocin- and kanamycin-resistant colonies were obtained from the electroporated cultures that had been induced with L-arabinose, while no colonies were obtained from the non-induced controls. Plasmid DNA isolated from individual clones was analyzed by restriction digest and basepair-precise modification of pFL98Q1 was confirmed by sequencing.
After each recombination step, analysis was performed on 6–12 clones. While 100% of the analyzed clones exhibited the expected fragments upon restriction analysis, additional bands indicated that all of the clones harbored mixtures of the target plasmid and the unmodified precursor plasmid (data not shown). This is a common observation when modifying multi-copy plasmids, since usually only very few plasmid copies in a cell undergo recombination. In order to separate recombined and unrecombined plasmid copies, 10 ng of plasmid DNA were retransformed into E. coli HS996. Retransformation clones harbored pure populations of the target plasmid as demonstrated by restriction digest (data not shown).
Plasmids pFL98Q2 and pFL98Q2-stop were subjected to restriction digest with SacII to release the fragments harboring full-length or truncated 98Q-htt cDNA and a downstream FRT-flanked kanamycin resistance cassette, flanked by 50 bp homology arms mediating insertion into the BDNF-BAC. The restriction fragments, 11.4 kb and 2.5 kb in size, were electroporated into E. coli cells harboring BAC RP23–106l19 and pRed/ET. ≥200 chloramphenicol- and kanamycin-resistant clones were obtained from the induced cultures. Twelve clones each were propagated and their BAC DNA was isolated and analyzed for correct integration of the htt cDNA by junction PCR. 100% of the analyzed clones (HTTstop-98Q) or 92% (11 out of 12) of the analyzed clones (FL-HTT-98Q) yielded the correct PCR signals (shown for FL-HTT-98Q clones in supplementary Figure 2A).
With a goal of generating BAC transgenes harboring full-length or truncated htt cDNA with a lower number of Q repeats (15Q) representing the genetic situation in healthy individuals free of HD, BACs RP23–106l19-FL-HTT-98Q and RP23–106l19-HTTstop-98Q were modified by counterselection using the rpsL gene (39). In a first Red/ET recombination step, an rpsL-zeocin counterselection cassette amplified by PCR with primers PCR4–5′ and PCR4–3′ (Supplementary Table 1) was integrated into BACs RP23–106l19-FL-HTT-98Q and RP23–106l19-HTTstop-98Q in place of the 98Q sequence. In a second Red/ET recombination step, the rpsL-zeocin cassette was replaced with a DNA fragment harboring 15Q repeats. The DNA fragment was obtained by PCR amplification from plasmid pIREScDNA15QHtt2, which harbors 15Q htt cDNA. Amplification was performed with primers PCR5–5′ and PCR5–3′ (Supplementary Table 1). After insertion of the rpsL-zeocin counterselection cassette, 12 zeocin-resistant clones each were confirmed by PCR across the junctions of the inserted rpsL-zeo cassette and were further confirmed to exhibit a streptomycin-sensitive phenotype. 100% of the analyzed clones yielded the expected amplification products (data not shown). Following replacement of the rpsL-zeo cassette, 12 streptomycin-resistant clones each were analyzed by PCR across the counterselection site. 100% of the analyzed clones yielded the expected PCR amplicon (shown for FL-HTT-98Q clones in Supplementary Figure 2B).