ABSTRACT
Laboratory mice are common experimental animals in biological, medical, pharmacological and psychological researches primarily because they are easy to maintain and reproduce quickly. The protection of the welfare of experimental animals is gaining greater attention during the application of a large number of mice. It's therefore essential to consider how to reduce the unnecessary use of animals and fully exploit each experimental animal. We report, in this article, an efficient way to dissect various brain regions from a mouse for protein immunoblot and/or neuronal culture, providing technical reference information for minimizing the number of animals used in projects, and refining methods and procedures to quick brain dissection.
Subject(s)
Animals , Mice , Animal Welfare , Brain , Dissection , MethodsABSTRACT
This study is aimed to clone and express human, rat alcohol dehydrogenase (ADH) and aldo-keto reductase. Then the enantioselective metabolism of mandelic acid (MA) was studied. Human alcohol dehydrogenase 2, rat alcohol dehydrogenase 1, human and rat aldo-keto reductase 1A1 were amplified using RT-PCR from human and rat liver samples. Then subcloned into pET-28a (+) and expressed in E. coli BL21 (DE3) stably. The protein was induced with IPTG and purified by affinity chromatography. Then the enzyme activities were measured. MA enantiomers were incubated with rat, human ADH and phenylglyoxylic acid (PGA) with AKR1A1, respectively. The metabolism was analyzed with HPLC. The proper genes were cloned and purified and proteins were obtained. All of the proteins obtained showed good activity. Stereoselective-metabolism of MA was observed in human ADH2, which favors for S-MA metabolism. The expression plasmids are constructed and the recombinant proteins are expressed successfully. The recombinant alcohol dehydrogenase and aldo-keto reductase have been employed to study MA metabolism.