High-level rearrangement and transcription of yeast artificial chromosome-based mouse Igκ transgenes containing distal regions of the contig

The mouse Igκ L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved. Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon Vκ-Jκ joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci. The largest minilocus (290 kb) possessed all members of the upstream Vκ2 gene family including their entire 5' and 3' flanking sequences, along with one member of a downstream Vκ21 gene family. In addition, again using yeast artificial chromosome-based technology, we created lgκ miniloci that contained differing lengths of sequences 5' of the most distal Vκ2 gene family member. In transgenic mice, Igκ miniloci exhibited position-independent and copy number-dependent germline transcription. Igκ miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal Vκ gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects. The minilocus that contained the longest 5' region exhibited significantly greater recombination of the upstream Vκ2 genes but not the downstream Vκ21 gene, providing evidence for a local recombination stimulating element. These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Igκ gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.