Identification of transcriptionally regulated mRNAs from mouse Schwann cell precursors using modified RNA fingerprinting methods.
We have adopted RNA fingerprinting methods to screen for genes that are rapidly up- or down-regulated during normal mammalian development, comparing mRNA from early (embryo day 12) to late (embryo day 13) mouse Schwann cell precursors. The use of total RNA, a reduction of cDNA template for amplification, the detection of RT-PCR products with a sensitive DNA stain and polyacrylamide gel electrophoresis and rigid selection criteria involving three screening steps are significant improvements on previous methods. Of 19 differentially displayed bands, 15 represented novel genes. The four known cDNA fragments (interleukin enhancer binding factor 1, beta3 subunit of phospholipase C, brain beta-spectrin, 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 were regulated between embryo days 12 and 13, as predicted by the display gels. Our results suggest that the combination of methods described here will have wide applicability in studies of other developmental systems where precisely timed changes occur and where only small amounts of RNA can be obtained for analysis.
Zoidl
G
G
Department of Anatomy and Developmental Biology, University College London, United Kingdom.
Blanchard
A D
AD
Zoidl
C
C
Dong
Z
Z
Brennan
A
A
Parmantier
E
E
Mirsky
R
R
Jessen
K R
KR
D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation; D018076:DNA, Complementary / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D005821:Genetic Techniques; D005865:Gestational Age; D051379:Mice; D008813:Mice, Inbred ICR; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009432:Neural Crest / Q000166:cytology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D012583:Schwann Cells / Q000166:cytology / Q000378:metabolism*; D012584:Sciatic Nerve / Q000166:cytology / Q000196:embryology; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D013382:Subtraction Technique; D014158:Transcription, Genetic
D015415:Biomarkers; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger