Stable transfer and expression of exogenous human globin genes in human erythroleukemia (K562) cells.
Stable transfer and expression of exogenous human globin genes in human erythroleukemia (K562) cells.
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外源人珠蛋白基因在人红白血病（K562）细胞中的稳定转移和表达。
DOI：
10.1073/pnas.81.17.5315
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发表时间：
1984
影响因子：
11.1
通讯作者：
Bank,A
中科院分区：
文献类型：
--
作者：
Young,K;Donovan-Peluso,M;Bloom,K;Allan,M;Paul,J;Bank,A
为了研究珠蛋白基因在人细胞中的表达，将人ε-珠蛋白基因转移到K562细胞系Bos中，其合成非常少量的ε-珠蛋白mRNA。将含有完整的ε-珠蛋白基因和5'侧翼DNA的2个内切酶（kb）以及新霉素抗性基因和猿猴病毒40复制起点的质粒（pSV 2neo-pSV）转染到Bos细胞中;使用化合物G418（一种新霉素类似物）来选择转化的细胞。通过DNA限制性分析的独特条带的存在表明，14个G418抗性克隆中的11个具有至少一个拷贝的整合的ε-珠蛋白基因。通过RNA印迹法测量的RNA表达显示出显着更多的ε-珠蛋白mRNA序列比未转染的Bos细胞中的10个11行;在大多数行中，ε-珠蛋白mRNA的存在下，氯化血红素另外增加。在两个线，ε-珠蛋白mRNA表达与氯化血红素是一个高ε-珠蛋白生产细胞系，K562克隆2。一个不含ε-珠蛋白基因的G418抗性品系没有ε-mRNA表达。在几个系中的高ε-mRNA表达表明，仅具有2-kb 5'侧翼DNA的外源ε-珠蛋白基因可能足以在这些同源红系细胞中适当表达。这些结果对将正常人基因转移到人骨髓细胞作为治疗遗传性贫血的一种方法的潜在成功具有意义。
To study the expression of globin genes in human cells, human epsilon-globin genes were transferred into a K562 cell line, Bos, which synthesizes very low amounts of epsilon-globin mRNA. A plasmid (pSV2neo-epsilon) containing a complete epsilon-globin gene and 2 kilobases (kb) of 5' flanking DNA as well as a neomycin-resistance gene and a simian virus 40 origin of replication was transfected into Bos cells; the compound G418, a neomycin analogue, was used to select transformed cells. The presence of unique bands by DNA restriction analysis shows that 11 of 14 of the G418-resistant clones have at least one copy of an integrated epsilon-globin gene. RNA expression measured by RNA blotting shows significantly more epsilon-globin mRNA sequences than in untransfected Bos cells in 10 of 11 lines; in most lines, epsilon-globin mRNA was additionally increased in the presence of hemin. In two lines, epsilon-globin mRNA expression with hemin was comparable to that of a high epsilon-globin producing cell line, K562 clone 2. The one G418-resistant line without epsilon-globin genes had no epsilon-mRNA expression. The high epsilon-mRNA expression in several of the lines suggests that exogenous epsilon-globin genes with only 2-kb 5' flanking DNA may be sufficient to be appropriately expressed in these homologous erythroid cells. These results have implications for the potential success of transfer of normal human genes to human bone marrow cells as an approach to the treatment of inherited anemias.