Abstract
BackgroundPromoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs). This approach requires modification and subcloning of BAC-DNA by recombineering technologies in Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction.FindingsIn this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp) were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination.ConclusionsThe time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb) fragments in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.
Highlights
Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs)
Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb) fragments in vivo without restriction and ligation. Both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains
Replacement of the Math5 open reading frame (ORF) using pKM208 To probe the potential of the plasmids to modify BACs, DH10B cells containing pKM208 and RP23-328P3 were transformed with a 4 kb CreERT2-Neo cassette with flanking homology arms of 708 bp and 372 bp (Figure 1A)
Summary
Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs). Bacterial artificial chromosomes (BACs) are low copy plasmids based on the F-plasmid of E. coli. They can carry up to 300 kb of foreign DNA. The large insert size has made BACs an important source for the generation of transgenic organisms, because they carry entire genes including their regulatory elements. This can be used to express foreign genes in a spatiotemporal pattern, which mimics endogenous gene expression [1,2,3,4]. BACs are propagated in recombination deficient E. coli strains to achieve high clonal stability and low rate of chimerism
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