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High-Level Rearrangement and Transcription of Yeast Artificial Chromosome-Based Mouse Ig Transgenes Containing Distal Regions of the Contig¹

Shuyu Li^*, Robert E. Hammer, Julia B. George-Raizen^*, Katherine C. Meyers^* and William T. Garrard^2,*

^* Department of Molecular Biology and Oncology and Biochemistry, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75235

The mouse Ig 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 V-J 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 V2 gene family including their entire 5' and 3' flanking sequences, along with one member of a downstream V21 gene family. In addition, again using yeast artificial chromosome-based technology, we created Ig miniloci that contained differing lengths of sequences 5' of the most distal V2 gene family member. In transgenic mice, Ig miniloci exhibited position-independent and copy number-dependent germline transcription. Ig miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal V 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 V2 genes but not the downstream V21 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 Ig gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.

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