Kerlakian C B, Toth S W, Kuempel E D, Luse D S
Mol Cell Biol. 1986 Nov;6(11):3873-83. doi: 10.1128/mcb.6.11.3873-3883.1986.
We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.
我们通过将小鼠β-珠蛋白主要启动子和初始转录区域与三种山羊β-样珠蛋白基因体之一融合,构建了三个杂合β-珠蛋白基因:βc(成年前)、βF(胎儿)或εII(胚胎)。胸苷激酶(tk)缺陷型小鼠红白血病(MEL)细胞与这些构建体之一和携带tk基因的单独质粒共转化。在含有小鼠β/山羊βc或小鼠β/山羊βF基因的24个细胞系中,有一半在显著水平上表达了转移基因;在许多情况下,杂合基因与内源性β-珠蛋白基因一样,可被二甲基亚砜诱导。我们获得了13个含有小鼠β/山羊εII杂合基因的细胞系,其中6个与已知在MEL细胞中起作用的小鼠β/人β融合基因共转染。与其他融合基因的结果相反,小鼠β/山羊εII杂合体的表达非常低:在两个独立实验中,诱导后13个细胞系中的0个和2个显示出显著的小鼠β/山羊εII RNA水平。在所有这些细胞系中,内源性小鼠β和共转染的小鼠β/人β基因均有表达。作为对小鼠β/山羊εII杂合体无活性可能原因的初步测试,我们将该融合基因重新克隆到一个携带tk的质粒中,与tk基因相邻。在用该质粒转化的12个细胞系中,11个产生了小鼠β/山羊εII RNA;在6个案例中,表达既强烈又可被二甲基亚砜诱导。