Abstract
BackgroundThe Bacillus subtilis genome (BGM) vector is a novel cloning system for large DNA fragments, in which the entire 4.2 Mb genome of B. subtilis functions as a vector. The BGM vector system has several attractive properties, such as a large cloning capacity of over 3 Mb, stable propagation of cloned DNA and various modification strategies using RecA-mediated homologous recombination. However, genetic modifications using the BGM vector system have not been fully established, and this system has not been applied to transgenesis. In this study, we developed important additions to the genetic modification methods of the BGM vector system. To explore the potential of the BGM vector, we focused on the fish-like odorant receptor (class I OR) gene family, which consists of 158 genes and forms a single gene cluster. Although a cis-acting locus control region is expected to regulate transcription, this has not yet been determined experimentally.ResultsUsing two contiguous bacterial artificial chromosome clones containing several class I OR genes, we constructed two transgenes in the BGM vector by inserting a reporter gene cassette into one class I OR gene. Because they were oriented in opposite directions, we performed an inversion modification to align their orientation and then fused them to enlarge the genomic structure. DNA sequencing revealed that no mutations occurred during gene manipulations with the BGM vector. We further demonstrated that the modified, reconstructed genomic DNA fragments could be used to generate transgenic mice. Transgenic mice carrying the enlarged transgene recapitulated the expression and axonal projection patterns of the target class I OR gene in the main olfactory system.ConclusionWe offer a complete genetic modification method for the BGM vector system, including insertion, deletion, inversion and fusion, to engineer genomic DNA fragments without any trace of modifications. In addition, we demonstrate that this system can be used for mouse transgenesis. Thus, the BGM vector system can be an alternative platform for engineering large DNA fragments in addition to conventional systems such as bacterial and yeast artificial chromosomes. Using this system, we provide the first experimental evidence of a cis-acting element for a class I OR gene.
Highlights
The Bacillus subtilis genome (BGM) vector is a novel cloning system for large DNA fragments, in which the entire 4.2 Mb genome of B. subtilis functions as a vector
Once a Bacterial artificial chromosome (BAC) insert is cloned into the BGM vector, the recombinants become resistant to Nm and sensitive to Spc, because the BAC insert replaces the cI-spc cassette
Once a BAC insert is cloned into the BGM vector, the recombinant becomes resistant to Nm and sensitive to Spc because the cI-spc cassette is replaced by the BAC insert. (B) Modifications of large DNA fragments
Summary
The Bacillus subtilis genome (BGM) vector is a novel cloning system for large DNA fragments, in which the entire 4.2 Mb genome of B. subtilis functions as a vector. Technological developments in chromosome engineering are essential for the manipulation and functional analysis of genomic DNA fragments. Artificial chromosomes, such as bacterial artificial chromosomes (BACs) [1] and yeast artificial chromosomes (YACs) [2], have to 2 Mb can be used with the YAC system. The isolation of intact YACs is difficult because of their linear form and contamination with endogenous yeast chromosomes These two systems have complementary advantages over each other in terms of cloning capacity and insert stability. Competent B. subtilis cells can be prepared by merely cultivating the cells in a special medium for several hours, and these cells are transformed by mixing in DNA fragments without additional heat-shock or electroporation [12]
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