Introduction of DNA fragments, hundreds of kilobases in size, into mouse embryonic stem (ES) cells would greatly advance the ability to manipulate the mouse genome. Mice generated from such modified cells would permit investigation of the function and expression of very large or crudely mapped genes. Large DNA molecules cloned into yeast artificial chromosomes (YACs) are stable and genetically manipulable within yeast, suggesting yeast-cell fusion as an ideal method for transferring large DNA segments into mammalian cells. Introduction of YACs into different cell types by this technique has been reported; however, the incorporation of yeast DNA along with the YAC has raised doubts as to whether ES cells, modified in this way, would be able to recolonize the mouse germ line. Here we provide, to our knowledge, the first demonstration of germ-line transmission and expression of a large human DNA fragment, introduced into ES cells by fusion with yeast spheroplasts. Proper development was not impaired by the cointegration of a large portion of the yeast genome with the YAC.
Using a recently described method for efficiently deriving homozygous targeted alleles in embryonic stem cells, we produced chimeric mice whose tissues were derived partially from embryonic stem cells bearing homozygous deletion of the mouse immunoglobulin heavy-chain joining (JH) region. Characterization of these chimeric mice indicated that homozygous JH deletion leads to arrest of B-cell development at an early stage, resulting in a total lack of peripheral B cells and serum IgM. These results were confirmed in mice containing the homozygous JH deletion in their germ line. This novel B-cell-deficient mouse strain provides a tool for studying the recombination and expression of exogenous immunoglobulin genes introduced into the mouse germ line.
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