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pubmed:abstractText |
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.
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