| Ornithine decarboxylase: Expression and regulation in rat brain and in transgenic mice | ||
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The transgenic mice were produced by the pronuclear microinjection technique (Hogan et al. 1986). Prior to microinjection, the pODC1.7 plasmid (II) was digested with Tth 111I¯ and BamHI to release the MMTV LTR promotor and ODC cDNA from the vector. The Tth111I - BamHI fragment was isolated by preparative agarose gel electrophoresis and purified by CsCl centrifugation. The purified DNA was injected into the pronucleus of fertilized oocytes from superovulated C57Bl/6 x C3H/Hej mice mated with males of the same hybrid strain. The microinjected zygotes were transferred into oviducts of pseudopregnant foster females.
The transgenic animals were identified by extracting high molecular weight DNA from 2 - 3 cm sections of mice tails. The primary detection of the transgene was carried out with the polymerase chain reaction and confirmed by Southern hybridization. The genotyped mice were analysed by determining ODC activity in tissue lysates and by histological examination of tissue sections. For ODC activity assays frozen tissues (kept at –70¢ªC) were homogenised in 1 – 1.5 ml of ice-cold 25 mM Tris-HCl, pH 7.4, containing 2.5 mM DTT, 0.1 mM PLP, 0.1 mM EDTA and 0.5 mM PMSF. The homogenates were clarified by centrifuging at 105 000 x g for 60 min at 4 ¢ªC. The supernatants were assayed for ODC enzyme activity. For histological examination fresh tissue samples were fixed in 4% formaldehyde in 10 mM sodium phosphate, 0.148 M NaCl, pH 7.4, and embedded in paraffin. Sections of 5 µm were cut, deparaffinized and stained with hematoxylin and eosin.