Isolation and Characterization of Murine Novel cDNA Clone Using Newly Constructed Gene Trap Vector.

Objective: To understand better the mechanism of the maintenance of immature state of stem cells, murine novel cDNA clones are to be isolated by the gene trap method.

Method: The trap vector was constructed with En-2 intron sequence followed by splicing acceptor sequence, IRES signal sequence and b-galactosidase cDNA. Neo^R gene was also contained without poly-A additional signal. After electroporation into Embryonic stem (Es) cell lines (D3, and E14.1 Köln), neomycin-resistant Es clones were picked up, and were selected in which the expression of b-gal was detected in the undifferentiated condition cultured with leukemia inhibitory factor (LIF), and feeder layer cells by X-gal staining, but were not detected in the differentiated condition with ATRA, and without LIF nor feeder layers. Using 5′ RACE method, the transcripts from the selected clones were identified. One clone was further analyzed. Full-length cDNA was isolated from a library constructed from Es D3 cells, and its characterization was determined.

Result and Discussion: one unique full-length cDNA clone was isolated for the further characterization. It was constructed with 2225 nucleotides, and putative 466 amino acids were encoded in a single long open reading frame. The transcript was detected in the undifferentiated Es cells but not in differentiated Es cells nor in various kinds of organs by northern blotting analysis. When this clone was over-expressed in murine 10T1/2 cells, RT-PCR analysis demonstrated the down-regulation of Id-1 gene. These data suggested that the isolated cDNA clone was possibly related to keeping Es cells to be immature multipotent state. We now try to isolate the related cDNA clones which are expressed mainly in the hematopoietic stem cells.