IDENTIFICATION OF TRANSCRIPTIONALLY REGULATED MESSENGER-RNAS FROM MOUSE SCHWANN-CELL PRECURSORS USING MODIFIED RNA FINGERPRINTING METHODS

We have adopted RNA fingerprinting methods to screen for genes that ar e rapidly up- or down-regulated during normal mammalian development, c omparing mRNA from early (embryo day 12) to late (embryo day 13) mouse Schwann cell precursors. The use of total RNA, a reduction of cDNA te mplate for amplification, the detection of RT-PCR products with a sens itive DNA stain and polyacrylamide gel electrophoresis and rigid selec tion criteria involving three screening steps are significant improvem ents on previous methods. Of 19 differentially displayed bands, 15 rep resented novel genes. The four known cDNA fragments (interleukin enhan cer binding factor 1, beta 3 subunit of phospholipase C, brain beta-sp ectrin, and P21 polypeptide) consisted of coding sequences. indicating a high chance of obtaining coding regions. A semiquantitative RT-PCR analysis of three of the four known genes and a cDNA fragment randomly selected from the pool of 15 novel sequences, confirmed that they wer e regulated between embryo days 12, and 13, as predicted by the displa y gels. Our results suggest that the combination of methods described here will have wide applicability in studies of other developmental sy stems where precisely timed changes occur and where only small amounts of RNA can be obtained for analysis. (C) 1997 Wiley-Liss, Inc.