Hendricks M B, Banker M J, McLaughlin M
Integrated Genetics, Inc., Framingham, MA 01701.
Gene. 1988 Apr 15;64(1):43-51. doi: 10.1016/0378-1119(88)90479-9.
We have constructed a high-efficiency vector for expression of genes of interest in myeloma cells. This vector is comprised of regulatory sequences from immunoglobulin (Ig) genes, including the heavy-chain enhancer, a kappa light-chain promoter and splice site, and the polyadenylation signal downstream from the kappa constant region. The expression capacity of this vector was assayed in J558L myeloma cells using human tissue plasminogen activator as a reporter gene. Stable transfectants were analyzed for protein, RNA, DNA copy number and transcription rate. Expression was compared to that of intact, transfected Ig genes and to endogenous Ig. Tissue plasminogen activator cloned into this vector was found to be expressed as efficiently as intact, transfected Ig genes, producing 1-2% of total cellular mRNA from a single copy of the gene. RNA levels and transcription rates relative to those of endogenous Ig genes were found to be about 25% and 38%, respectively. Because of its high efficiency, potential for gene amplification, and various scale-up advantages of myeloma cells, this vector-host system may yield levels of desired proteins than currently available systems.
我们构建了一种用于在骨髓瘤细胞中表达目的基因的高效载体。该载体由免疫球蛋白（Ig）基因的调控序列组成，包括重链增强子、κ轻链启动子和剪接位点，以及κ恒定区下游的聚腺苷酸化信号。使用人组织纤溶酶原激活剂作为报告基因，在J558L骨髓瘤细胞中检测了该载体的表达能力。对稳定转染子进行了蛋白质、RNA、DNA拷贝数和转录率分析。将表达与完整的、转染的Ig基因以及内源性Ig的表达进行了比较。发现克隆到该载体中的组织纤溶酶原激活剂与完整的、转染的Ig基因一样高效表达，从该基因的单拷贝产生占总细胞mRNA 1-2%的产物。相对于内源性Ig基因，RNA水平和转录率分别约为25%和38%。由于其高效性、基因扩增潜力以及骨髓瘤细胞的各种放大优势，该载体-宿主系统可能产生比现有系统更高水平的所需蛋白质。
