Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells
| Autor: | Catherine E. Maynard-Currie, Hirohisa Tsuda, Dan Garza, Nathaniel E. David, Michael Mendez, Robert M. Gemmill, Larry L. Green, Sue Klapholz, Aya Jakobovits, Sienna Yoast, Margaret C. Hardy, Hadi Abderrahim, Masato Noguchi |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Yeast artificial chromosome
Hypoxanthine Phosphoribosyltransferase congenital hereditary and neonatal diseases and abnormalities Molecular Sequence Data DNA Recombinant Immunoglobulin Variable Region chemical and pharmacologic phenomena Saccharomyces cerevisiae Gene mutation Biology Molecular cloning Immunoglobulin light chain Polymerase Chain Reaction Cell Fusion Immunoglobulin kappa-Chains Mice hemic and lymphatic diseases Gene cluster Genetics Animals Humans Cloning Molecular Selection Genetic Enhancer Chromosomes Artificial Yeast Gene Selectable marker Gene Library B-Lymphocytes Base Sequence Genes Immunoglobulin Stem Cells hemic and immune systems Fibroblasts Embryo Mammalian Molecular biology Immunoglobulin J-Chains Immunoglobulin Constant Regions Immunoglobulin Heavy Chains |
| Zdroj: | Genomics. 26:294-307 |
| ISSN: | 0888-7543 |
| DOI: | 10.1016/0888-7543(95)80214-7 |
| Popis: | 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 (V H ) genes, the major diversity (D) gene cluster, the joining (J H ) genes, the intronic enhancer (E H ), and the constant region genes, mu (C μ ) and delta (C δ ). Two IgK locus-derived YACs each contain three variable (V κ ) genes, the joining (J κ ) region, the intronic enhancer (E κ ), the constant gene (C κ ), 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. |
| Databáze: | OpenAIRE |
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