Yiwen Zhu, Miles Miller, Jan-Fang Cheng, and Eddy Rubin
Human Genome Center, Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
The interleukin gene cluster on human chromosome 5q31 was selected for large scale sequencing because it contains a number of functionally related genes and is therefore likely to yield novel cytokine gene candidates through sequence analysis. Parallel to sequencing and sequence analysis, we have begun to develop a mouse model to study functions of the sequenced genes.
Saturated mutation of a defined region in the mouse genome has only recently become feasible with the very recent development of a new genetic engineering technique, the Cre-Lox site-specific recombination. It is based on the ability of Cre recombinase to delete regions bound by loxP sites (a 34-bp sequence) in vivo. Application of such technology to mouse embryonic stem (ES) cells would allow large chromosome deletions to be introduced into the germline and therefore would facilitate new functional analyses of the genome. The size of deletions could be multimegabases since translocation between mouse chromosomes has been previously reported for the Cre-Lox recombination. Multimegabase deletions, however, frequently produce an embryonic lethal phenotype. One key advantage of the Cre-Lox approach is the ability to generate deletions specific to a developmental stage and tissue. LoxP targeted ES cells in which the deletion has not yet been activated would be used to achieve germline transmission. The deletion could then be induced by crossing ES cell-derived progeny with transgenic strains in which Cre recombinase expression is under control of a developmental stage-specific and tissue-specific promotor.
In our targeting experiments, loxP sites were inserted into the murine homologue of the human 5q31 region. The availability of previously cloned and sequenced genes (IL4, IL13, IL5, IRFI, CSF2, IL3) dispersed in the targeted region of mouse chromosome 11 provided the required sequence information for creating targeting vectors. Four targeting vectors carrying loxP sequences and either neomycin (Neo[r]) or hygromycin (Hyg[r]) markers were designed to integrate into 4 locations in the gene cluster. These locations are (1) 5' flank of the IL13 gene, (2) 10.5 Kb upstream of the IL5 gene, (3) internal of the IRF1 gene, and (4) 3' flank of the CSF2 gene. All 4 targeting vectors were successfully inserted into the predetermined sites. We have confirmed the targeted integrations using restriction digests and Southern blot analysis of the ES cell genomic DNA. The frequency of appropriated targeting event varied from 46% to 4%.
We are now in the process of injecting targeted ES cells into mouse embryos. This will allow the production of mouse strains in which all the cells contain the targeted DNA. These animals will be bred with animals expressing Cre recombinase in either all or selected tissues (such as lymphoid) to create a series of nested deletions. The progeny of these matings will be characterized phenotypically to identify specific sequences whose absence impacts on the development of certain cell lineages in the immune system. This in vivo analysis combined with 1.2 Mb of sequence data from human 5q31 will contribute to the discovery and functional analysis of genes contained within this targeted region of the human genome.
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