cDNA cloning, characterization, expression and recombinant protein production of leukemia inhibitory factor (LIF) from the marsupial, the brushtail possum (Trichosurus vulpecula).

ABSTRACT

A reverse transcription technique using RNA templates combined with polymerase chain reaction (RT-PCR) was used to clone the cDNA fragment encoding the amino acid sequence of mature LIF protein of the marsupial, the brushtail possum, Trichosurus vulpecula. A PCR product with expected size, of 546bp, and termed tvLIF, was obtained using cDNA reverse-transcribed from total RNA isolated from possum uterus. A genomic DNA fragment (about 650bp) between the specified primers was also amplified, indicating the similarity in structure and organization of this gene and LIF genes from studied eutherian species, although the full-length of its cDNA and genomic DNA needs to be further clarified. The deduced amino acid sequence of tvLIF shows a high level of sequence identity and similar molecular characteristics to eutherian LIF, which suggests similar biological actions of this molecule in this marsupial. Because the expression of LIF gene in other mammalian species has been found to be at very low levels and its transcripts cannot be detected by Northern hybridization analysis, the expression pattern of tvLIF in adult tissues and reproductive tracts during early development was investigated using the RT-PCR technique. Resultant products of the RT-PCR were further analyzed by Southern hybridization using tvLIF as a probe. tvLIF transcripts were detected in most of the adult tissues and in the reproductive tracts of pregnant females. These results lend support to the idea that LIF contributes to the maintenance of pregnancy in this marsupial.