A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus

The mouse Ig H chain (Igh) complex locus is composed of >100 gene segments encoding the variable, diversity, joining, and constant portions of the Ab H chain protein. To advance the characterization of this locus and to identify all the V(H) genes, we have isolated the entire region from C57BL/6 and C57BL/10 as a yeast artificial chromosome contig. The mouse Igh locus extends approximately three megabases and contains at least 134 V(H) genes classified in 15 partially interspersed families. Two non-Igh pseudogenes (Odc-rs8 and Rpl32-rs14) were localized in the distal part of the locus. This physical yeast artificial chromosome map will provide important structure and guidance for the sequencing of this large, complex, and highly repetitive locus.     

Evolution of a 6-200 Mb long-range repeat cluster in the genus Mus

By fluorescence in situ hybridization, we mapped the location of genes associated with the Sp100-rs cluster, a long-range repeat cluster in chromosome 1 of the house mouse, Mus musculus. The cluster comprises between 60 and 2000 repeats and extends over 6-200 Mb of the M. musculus genome, depending on the source of the cluster. The cluster evolved during the last two million years in the genus Mus in the lineage to which M. musculus belongs. The Asiatic mouse species M. caroli is not in this lineage and does not possess the cluster. M. caroli represents the ancestral genomic organization of the cluster source components Sp100, Csprs and Ifi75: they are located close to each other in the same chromosome band (1D). However, Sp100-rs, the principal gene of the cluster, is not present in the M. caroli genome. It is a chimeric M. musculus gene that arose by fusion of Csprs and the 5' part of Sp100. Sp100-rs and Ifi75 are homogeneously distributed throughout the cluster while Sp100 and Csprs in its original sequence context flank the cluster on opposite sides. Our results suggest a model for the origin and evolution of the long-range repeat cluster by duplication, gene fusion and amplification.     

Mus Spretus Line-1 Sequences Detected in the Mus Musculus Inbred Strain C57bl/6j Using Line-1 DNA Probes

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.     

Yeast artificial chromosome targeting technology: an approach for the deletion of genes in the C57BL/6 mouse

An approach is described to modify yeast artificial chromosomes (YACs) with cassettes that can be easily excised for embryonic stem (ES) cell gene targeting experiments. YAC targeting technology (YTT) uses the WIBR/MIT-820 C57BL/6-mapped YAC library derived from the C57BL/6 mouse as the starting point for Internet- or PCR-based clone isolation, although in principle any YAC system can be used. Homologous recombination is initially performed in yeast using cassettes that function in Saccharomyces cerevisiae, Escherichia coli, and ES cells, followed by cloning or conversion of the targeted locus into a plasmid. The completed targeting vector can be transfected into C57BL/6 ES cells and clones selected with G418 followed by injection into Balb/c blastocysts. YTT increases the speed of targeting vector construction and obviates the need for extensive backcrossing to the C57BL/6 background.     

Expression of mouse::human immunoglobulin heavy-chain cDNA in lymphoid cells

A chimeric mouse variable::human constant immunoglobulin heavy-chain gene was expressed in transfected mouse Sp2/0 cells. The chimeric immunoglobulin genes were integrated in tandem in the genome of stably transformed cells. These integrated gene copies were amplified by selection with a second drug marker. The gene amplification led to an increase in the expression of chimeric heavy-chain protein. The level of gene expression appears to be related to the site of integration; a few gene copies in one transfectant can yield as much heavy-chain protein as many copies in a second transfectant. In addition, we found that an adventitious oligo(C) sequence, introduced by our method of gene construction at a site located 8 nt residues downstream from a splice acceptor, can apparently direct splicing towards a cryptic splice acceptor downstream from the oligo(C).