Shen Q, van Beusechem V W, Einerhand M P, Hendrikx P J, Valerio D
Institute of Applied Radiobiology and Immunology-TNO, Rijswijk, The Netherlands.
Gene. 1991 Feb 15;98(2):283-7. doi: 10.1016/0378-1119(91)90187-g.
A eukaryotic expression vector was constructed in which the coding nucleotide sequences (ADA) of human adenosine deaminase (ADA) were fused in frame with the coding sequences of the bacterial gene lacZ encoding beta-galactosidase (beta Gal). This ADA::lacZ fusion gene was anticipated to encode a hybrid protein that has retained the biological functions of both proteins. Transfection of mammalian cells with the fusion gene resulted in the synthesis of both ADA and beta Gal. Cells expressing the gene could therefore be detected with the histochemical staining procedure that relies on the conversion of the indicator, XGal, by beta Gal. In addition, the transfected cells could be sorted on a fluorescence-activated cell sorter with the use of a vital staining procedure described for the selection of beta Gal-producing cells. Cell lines that harbored the fusion gene were tested for ADA overexpression by exposing them to the cytotoxic adenosine analog 9-beta-D-xylofuranosyl adenine (Xyl-A), in the presence of the ADA inhibitor deoxycoformycin (dCF). Resistance to Xyl-A/dCF was observed in the lines carrying ADA::lacZ and moreover, the fraction of cells that survived a stringent selection for ADA overexpression also exhibited significantly increased levels of beta Gal, which confirmed the direct linkage between ADA and lacZ expression. The use of this and other fusion genes might be useful in the development of gene-therapy protocols where they could help to meet the demand for versatile methods to detect and select cells with newly introduced genes.
构建了一种真核表达载体，其中人腺苷脱氨酶（ADA）的编码核苷酸序列（ADA）与编码β-半乳糖苷酶（βGal）的细菌基因lacZ的编码序列读框融合。预计该ADA::lacZ融合基因可编码一种保留了两种蛋白质生物学功能的杂合蛋白。用融合基因转染哺乳动物细胞可导致ADA和βGal的合成。因此，可通过依赖βGal对指示剂XGal进行转化的组织化学染色程序来检测表达该基因的细胞。此外，可使用为选择产生βGal的细胞而描述的活细胞染色程序，在荧光激活细胞分选仪上对转染细胞进行分选。通过将携带融合基因的细胞系暴露于细胞毒性腺苷类似物9-β-D-木糖呋喃基腺嘌呤（Xyl-A），并在ADA抑制剂脱氧助间霉素（dCF）存在的情况下，测试其ADA过表达情况。在携带ADA::lacZ的细胞系中观察到对Xyl-A/dCF的抗性，而且，在经过严格筛选ADA过表达后存活的细胞部分也显示出βGal水平显著增加，这证实了ADA与lacZ表达之间的直接联系。使用这种融合基因和其他融合基因可能有助于开发基因治疗方案，在这些方案中，它们可有助于满足对检测和选择具有新导入基因的细胞的通用方法的需求。
