|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
It is known that the formation of highly reactive dicarbonyl compounds such as 3-deoxyglucosone (3-DG), an intermediate of the glycation reaction, is accelerated in diabetic subjects. Several lines of evidence have shown that excess 3-DG influences cell functions in vitro, suggesting its involvement in the development of diabetic microangiopathy. On the other hand, it has been proposed that there is a defense system against 3-DG by metabolizing it to an inert agent in vivo. In this regard, previous studies have shown that aldehyde reductase (ALR) plays a major role in it. Thus, the present study was designed to clarify the relationship between 3-DG metabolism and alteration in microvascular tissues by establishing ALR-knockout mice. We first prepared a probe to clone mouse ALR cDNA by conducting PCR on mouse liver cDNA library with a set of primer deduced from the known rat ALR cDNA sequence. Using this probe, we successfully cloned a full length of mALR cDNA in 1999. We subsequently obtained a genome ALR DNA from a mouse genome DNA library with a probe including the start codon, followed by analysis of its sequence. We then constructed a targeting vector aiming homologous replacement in its exon 2〜5. Simultaneously, we prepared certain probes to subsequently screen gene-targeted ES cells by Southern blotting among the transfected cells with the vector described above.
We are going to microinject the ALR-gene-targeted ES cells into early embryo for generating chimeras, followed by establishing knockout mice. Finally, the mice were analyzed in terms of the relationship between 3-DG metabolism and alterations occurring in microvasculature tissues.