생쥐의 뇌 발현에 관여하는 5 -UTR 도서관의 제조

Construction of 5 -Upstream Region Library Controlling Brain Expression in Mouse

We are constructed 5 -upstream region(UTR) library using mRNAs isolated from mouse brain. To exclude mRNA pool with the damage in the 5 -ends most of which don t include 5 -UTR, the mRNA pool is treated with bacterial alkaline phosphatase and tobacco acid pyrophosphstase. The two enzymes make the intact mRNAs have a phosphate group in the 5 -ends, while the damaged mRNAs possess hydroxy group in the 5 -ends of them. Consequently, only intact mRNAs including 5 -UTRs can attach a XB RNA linker to 5 -ends of them. First-stranded cDNAs synthesized by using ATG primers are amplifed from PCR with the ATG primers and the XB primers. The amplified PCR products, which contain 5 -UTR, are subjected to cloning to lambda DNA vector. The titer of constructed 5 -UTR library is 2.75 x 105 pfu/mL. To investigate the efficiency of the library, twenty eight clones are chosen at random and sequenced. By analysis of their sequences, it s efficiency is approximately 40% and the average size of 5 -UTRs is 132 bp, implying that the quality of the library is as good as it can be used to isolate promoters for genes expressed in moue brain.