Rajagopalan L E, Burkholder J K, Turner J, Culp J, Yang N S, Malter J S
Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison 53792-2472, USA.
Blood. 1995 Oct 1;86(7):2551-8.
To increase transgenic production of granulocyte-macrophage colony-stimulating factor (GM-CSF), we mutated the mRNA's 3'-untranslated region, AUUUA instability elements. Expression vectors containing human or murine GM-CSF cDNAs coding for wild-type (GM-AUUUA) or mutant versions with reiterated AUGUA repeats (GM-AUGUA) were transfected into cells in culture or animals using particle-mediated gene-transfer technology. Normal peripheral blood mononuclear cells accumulated 20-fold greater levels of GM-CSF mRNA and secreted comparably greater amounts of cytokine after transfection with hGM-AUGUA expression vectors versus hGM-AUUUA. hGM-AUGUA mRNA was fivefold more stable (t 1/2 = 95 minutes) than hGM-AUUUA mRNA (t 1/2 = 20 minutes), accounting for elevated steady-state levels. Transfection site extracts and serum samples obtained 24 hours after gene transfer of hGM-AUGUA cDNA into mouse skin contained greater than 32 ng/mL and 650 pg/mL of GM-CSF protein, respectively, compared with 0.33 ng/mL and less than 8 pg/mL for hGM-AUUUA cDNA. GM-CSF produced from mGM-AUGUA cDNA transfected into rat abdominal epidermis induced a profound neutrophil infiltrate. These data suggest a novel strategy for enhanced production of biologically active cytokines by normal cells after in vivo gene transfer.
为提高粒细胞-巨噬细胞集落刺激因子（GM-CSF）的转基因产量，我们对mRNA的3'-非翻译区，即AUUUA不稳定元件进行了突变。使用粒子介导的基因转移技术，将含有编码野生型（GM-AUUUA）或带有重复AUGUA重复序列的突变体版本（GM-AUGUA）的人或小鼠GM-CSF cDNA的表达载体转染到培养细胞或动物中。与hGM-AUUUA相比，用hGM-AUGUA表达载体转染后，正常外周血单核细胞积累的GM-CSF mRNA水平高20倍，分泌的细胞因子量也相应增加。hGM-AUGUA mRNA的稳定性比hGM-AUUUA mRNA高5倍（半衰期=95分钟），而hGM-AUUUA mRNA的半衰期为20分钟，这解释了其稳态水平升高的原因。将hGM-AUGUA cDNA基因转移到小鼠皮肤24小时后获得的转染部位提取物和血清样本中，GM-CSF蛋白含量分别大于32 ng/mL和650 pg/mL，而hGM-AUUUA cDNA的含量分别为0.33 ng/mL和小于8 pg/mL。转染mGM-AUGUA cDNA到大鼠腹部表皮产生的GM-CSF诱导了大量中性粒细胞浸润。这些数据表明了一种通过体内基因转移后正常细胞增强生物活性细胞因子产生的新策略。
