Difference between revisions of "Serine Racemase"
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| − | + | =Regulation of SR gene expression= | |
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There are two approaches to examining changes in SR expression in vivo: by pharmacological manipulaton, and by characterization across development. | There are two approaches to examining changes in SR expression in vivo: by pharmacological manipulaton, and by characterization across development. | ||
| − | + | ==Pharmacological Manipulations== | |
| − | <b>MK-801: < | + | <b>MK-801: </b> Hasimoto et al. PMID 17109841 used qRT-PCR and HPLC to detect changes in SR and DAAO in adult rat brain after acute or chronic injections of MK-801, the non-competitive NR antagonist. Changes in mRNA were only seen at 400 ug/kg and above, Very large changes (5x - 10x at 1 h, still elevated 2-3x at 4 h) were seen in all brain regions examined (striatum hippocampys, cortex, diencephalon, midbrain, pons/medulla and cerebellum (although absolute levels were lower in more caudal brain regions.) DAAO mRNA levels were decreased by MK-801 at 1 h, but increased at 4 h (and absolute levels of DAAO mRNA were high in more caudal brain regions.) DAAO showed a more dose-dependent response. Chronic MK-801 (400 ug/kg for 14 days) also increased SR mRNA (2x in forebrain) but did not affect DAAO expression. There is little discussion of the possible mechanisms, aside from pointing out that MK-801 can induce c-fos and there is an AP-1 element in the SR promoter, and a CRE element in the DAAO promoter. |
| − | + | =Probe for in situ hybridization= | |
Primer sequences taken from Yoshikawa et al., PMID 16973158 | Primer sequences taken from Yoshikawa et al., PMID 16973158 | ||
Revision as of 13:34, 20 February 2007
Regulation of SR gene expression
There are two approaches to examining changes in SR expression in vivo: by pharmacological manipulaton, and by characterization across development.
Pharmacological Manipulations
MK-801: Hasimoto et al. PMID 17109841 used qRT-PCR and HPLC to detect changes in SR and DAAO in adult rat brain after acute or chronic injections of MK-801, the non-competitive NR antagonist. Changes in mRNA were only seen at 400 ug/kg and above, Very large changes (5x - 10x at 1 h, still elevated 2-3x at 4 h) were seen in all brain regions examined (striatum hippocampys, cortex, diencephalon, midbrain, pons/medulla and cerebellum (although absolute levels were lower in more caudal brain regions.) DAAO mRNA levels were decreased by MK-801 at 1 h, but increased at 4 h (and absolute levels of DAAO mRNA were high in more caudal brain regions.) DAAO showed a more dose-dependent response. Chronic MK-801 (400 ug/kg for 14 days) also increased SR mRNA (2x in forebrain) but did not affect DAAO expression. There is little discussion of the possible mechanisms, aside from pointing out that MK-801 can induce c-fos and there is an AP-1 element in the SR promoter, and a CRE element in the DAAO promoter.
Probe for in situ hybridization
Primer sequences taken from Yoshikawa et al., PMID 16973158
Rat serine racemase. Genbank Ascension # NM_198757
PCR product Length: 295 bases (+22 bases for T7 promoter]
Forward Primer 664 to 683
- ATT GCA AGA AAC TGG CCA TC
Reverse Primer [with T7 promoter] 958 to 939
- [CTT AAT ACG ACT CAC TAT AGG G] TC AGC AGC GTA CAC CTT CAC
Amplification conditions (from Yoshikawa et al, PMID 15337321):
- 10-min predenaturation at 95 °C
- 15-s denaturation at 95 °C.
- 20-s at 58 °C for serine racemase
- followed by a 20-s extension at 72 °C.