BLASTICIDIN-S DEAMINASE GENE FROM ASPERGILLUS-TERREUS (BSD) - A NEW DRUG-RESISTANCE GENE FOR TRANSFECTION OF MAMMALIAN-CELLS
BLASTICIDIN-S DEAMINASE GENE FROM ASPERGILLUS-TERREUS (BSD) - A NEW DRUG-RESISTANCE GENE FOR TRANSFECTION OF MAMMALIAN-CELLS
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DOI：
10.1016/0167-4781(94)90224-0
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发表时间：
1994-11-22
期刊：
影响因子：
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通讯作者：
YAMAGUCHI, I
中科院分区：
文献类型：
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作者：
KIMURA, M;TAKATSUKI, A;YAMAGUCHI, I
杀稻瘟菌素S脱氨酶（BSD）是由土曲霉产生的一种药物失活酶，可将杀稻瘟菌素S（BS）转化为无毒的脱氨基羟基衍生物。将BSD基因与SV 40转录调控元件融合，并将所得载体用于转染FM 3A细胞。BSD的表达赋予了对BS的抗性，并允许在重复转移到新鲜选择培养基后稳定保持BS抗性表型的整合转染子的有效分离。转染的频率与neo相当，并且比细菌来源的BS抗性基因bsr高约80倍，该细菌来源的BS抗性基因可用于有效地分离转染的HeLa细胞。利用BSD作为选择标记，我们获得了几个稳定表达萤火虫荧光素酶基因的细胞系。在随机选择的5个BS抗性菌落中，4个独立的转染子在表达载体中的地塞米松诱导型启动子的控制下表现出可检测的荧光素酶活性。BSD的成功应用有力地表明了BS作为一种多功能选择性试剂用于将克隆的DNA序列导入哺乳动物细胞的有用性。
Blasticidin S deaminase (BSD) is a drug inactivating enzyme produced by Aspergillus terreus, which convert blasticidin S (BS) to a non-toxic deamino-hydroxy derivative. The BSD gene was fused to SV 40 transcriptional regulatory elements and the resulting vector was used to transfect FM3A cells. Expression of BSD conferred resistance to BS and allowed efficient isolation of integrative transfectants which have stably maintained the BS-resistance phenotype after repeated transfer to fresh selective medium. The frequency of transfection was comparable to that with neo and about 80-times greater than with bsr, a BS-resistance gene of bacterial origin which can be used to isolate efficiently transfectant HeLa cells. Using BSD as a selectable marker, we obtained several stable cell lines expressing the firefly luciferase gene. Four independent transfectants among the randomly selected 5 BS-resistance colonies exhibited detectable luciferase activity under the control of dexamethasone-inducible promoter in the expression vector. The successful application of BSD strongly suggests the usefulness of BS as a versatile selective reagent for introduction of cloned DNA sequences into mammalian cells.