Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells

Michael J. Mendez, Hadi Abderrahim, Masato Noguchi, Nathaniel E. David, Margaret C. Hardy, Larry L. Green, Hirohisa Tsuda, Sienna Yoast, Catherine E. Maynard-Currie, Dan Garza, Robert Gemmill, Aya Jakobovits, Sue Klapholz

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Abstract

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (Cμ) and delta (Cδ). Two IgK locus-derived YACs each contain three variable (Vκ) genes, the joining (Jκ) region, the intronic enhancer (Eκ), the constant gene (Cκ), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

Original languageEnglish
Pages (from-to)294-307
Number of pages14
JournalGenomics
Volume26
Issue number2
Publication statusPublished - 20 Mar 1995
Externally publishedYes

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ASJC Scopus subject areas

  • Genetics

Cite this

Mendez, M. J., Abderrahim, H., Noguchi, M., David, N. E., Hardy, M. C., Green, L. L., Tsuda, H., Yoast, S., Maynard-Currie, C. E., Garza, D., Gemmill, R., Jakobovits, A., & Klapholz, S. (1995). Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells. Genomics, 26(2), 294-307.