Uberla K, Li W Q, Qin Z H, Richter G, Raabe T, Diamantstein T, Blankenstein T
Institut für Immunologie, Universitätsklinikum Steglitz, Freie Universität Berlin, Germany.
Cytokine. 1991 Jan;3(1):72-81. doi: 10.1016/1043-4666(91)90012-3.
The rat interleukin-5 (IL-5) gene was isolated from a genomic lambda phage library and a fragment containing all four exons was inserted into the retroviral vector pXT1, resulting in pXTRIL5. Upon retroviral gene transfer into two IL-5-dependent mouse cell lines, B13 and T88M, autonomously growing cells were established and B-cell growth factor activity was detected in the supernatants of the infected cells. "cDNA" versions of the rat IL-5 gene were rescued by the polymerase chain reaction (PCR) with primers specific for the flanking regions of the cloning site in pXT1. Restriction or DNA sequence analysis of five different clones revealed precise splicing in two cases, while three of the clones had retained the first intron. In addition, in two of these about 400 bp of rat IL-5 5' flanking regions were deleted. The sequence comparison of rat, mouse, and human IL-5 genes revealed a high degree of conservation (e.g., mouse and rat were 92% homologous at the amino acid level). The combination of retroviral gene transfer and PCR may offer an alternative, efficient method for the cloning of cDNAs.
从基因组λ噬菌体文库中分离出大鼠白细胞介素-5（IL-5）基因，并将包含所有四个外显子的片段插入逆转录病毒载体pXT1中，得到pXTRIL5。将逆转录病毒基因导入两种依赖IL-5的小鼠细胞系B13和T88M后，建立了自主生长的细胞系，并在感染细胞的上清液中检测到B细胞生长因子活性。用针对pXT1中克隆位点侧翼区域的特异性引物，通过聚合酶链反应（PCR）获得大鼠IL-5基因的“cDNA”版本。对五个不同克隆进行的限制性酶切或DNA序列分析显示，其中两个克隆发生了精确剪接，而另外三个克隆保留了第一个内含子。此外，在其中两个克隆中，约400 bp的大鼠IL-5 5'侧翼区域被缺失。大鼠、小鼠和人IL-5基因的序列比较显示出高度保守性（例如，小鼠和大鼠在氨基酸水平上的同源性为92%）。逆转录病毒基因转移和PCR相结合可能为cDNA克隆提供一种替代的高效方法。
