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
Cell Genesys, Inc., Foster City, California 94404, USA.
Genomics. 1995 Mar 20;26(2):294-307. doi: 10.1016/0888-7543(95)80214-7.
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 (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), 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.
为了培育出能够产生人类抗体的小鼠品系，我们正在酵母人工染色体（YAC）中克隆和重建人类免疫球蛋白种系库。我们描述了从人类重链（IgH）和κ轻链（IgK）基因座中鉴定出包含可变区和恒定区序列的YAC，以及它们在酵母和小鼠胚胎干细胞（ES细胞）中的完整性特征。源自IgH基因座的YAC包含五个可变（VH）基因、主要多样性（D）基因簇、连接（JH）基因、内含子增强子（EH）以及恒定区基因μ（Cμ）和δ（Cδ）。两个源自IgK基因座的YAC各自包含三个可变（Vκ）基因、连接（Jκ）区域、内含子增强子（Eκ）、恒定基因（Cκ）以及κ缺失元件（kde）。IgH YAC在酵母中不稳定，可以产生多种缺失衍生物，而两个IgK YAC都是稳定的。将携带哺乳动物选择标记次黄嘌呤磷酸核糖转移酶（HPRT）的编码重链和κ轻链的YAC分别导入缺乏HPRT的小鼠ES细胞中。对ES细胞系中YAC完整性的分析表明，大多数DNA插入片段以基本完整的形式整合。
