mRNA differential display of acute-phase proteins in experimental Escherichia coli infection.
We present a modification of mRNA differential display in which increased throughput results from the use of an automated fluorescent sequencer. The sequence analysis is performed directly on purified fragments without further cloning. The amplified fragments carry a T7 RNA polymerase promoter sequence tag for in vitro transcription of riboprobes for nonradioactive in situ hybridization. We compared changes in gene expression in the liver and colon of group II phospholipase A2 transgenic and group II phospholipase A2 deficient mice during the course of experimental Escherichia coli infection. Fluorescent mRNA differential display comprising a 7 x 24 set of primers was used to study a total of 31,257 amplified cDNA fragments. Sequence analysis of the displayed fragments associated with infection identified classical acute-phase proteins in the liver and host defense proteins in the colon. The displayed mRNAs associated to transgenicity were the transgene itself, i.e., human group II phospholipase A2, and glutathione-S-transferase in the liver. In the colon, the displayed mRNAs associated with transgenicity were the pancreatitis-associated protein and mucin. The results show that fluorescent mRNA differential display is a reliable method to identify differences in the expression of the genes of acute-phase proteins.
Ojala
P
P
Department of Pathology, University of Turku, Finland.
Laine
V J
VJ
Raunio
J
J
Grass
D S
DS
Nevalainen
T J
TJ
D000209:Acute-Phase Proteins / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D004586:Electrophoresis / Q000379:methods; D004927:Escherichia coli Infections / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D009928:Organ Specificity; D000073718:Pancreatitis-Associated Proteins; D016133:Polymerase Chain Reaction / Q000379:methods
D000209:Acute-Phase Proteins; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human