Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse livers of adult and newborn animal response to Aflatoxin B1

ABSTRACT: Susceptibility to the hepatocarcinogen Aflatoxin B1 (AFB1) varies among species and with age. Mice are refractory to carcinogenic and toxic effects of AFB1; however, B6C3F1 mice show transient sensitivity if dosed shortly after birth. We compared age-related differences in gene expression and transcriptional responses to AFB1 in livers of newborn (4-day-old) and adult mice. Experiment Overall Design: Experiments were carried out under the protocol approved by MIT Animal Care Committee. Pregnant C57BL/6J female mice, mated to C3H/HeJ males, and adult (at least 60 day-old) B6C3F1/J hybrid F1 male mice (C57BL/6J female x C3H/HeJ male) were obtained from Jackson Labs. They were housed in plastic cages (one per cage) under controlled environmental conditions and 12 h light/dark cycle. Food and water were supplied ad libitum. After female mice gave birth, the male B6C3F1 pups were dosed at 4 days by a single i.p. injection of AFB1 (6 mg/kg body weight) in DMSO (10 ml/kg body weight) or the same volume of just DMSO. The treatments were always performed at the same time of the day and the pups were returned to their mothers until they were sacrificed 4 or 24 h after treatment. Adult B6C3F1 males were treated with the same doses and volumes of AFB1 or DMSO after an acclimation period of at least two weeks and sacrificed 4 or 24 hours after treatment. We noticed that injection of a volume of 10 ml/kg (whether AFB1 or vehicle alone) is tolerated poorly by adult animals and thus another group of adult animals was added which was treated with the same (6 mg/kg) dose of AFB in lower (5 ml/kg) volume. Corresponding vehicle control animals (5ml/kg DMSO) were also used. In addition, untreated controls were used and sacrificed together with the treated animals. Three independant bilogical replicates were used for each experimental point. Total RNA was isolated from livers and processed according to standard Affymetrix protocol.

ORGANISM(S): Mus musculus

SUBMITTER: Yuriy Alekseyev

PROVIDER: E-GEOD-4740 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To reveal the comparative pharmacological mechanism of differential compounds and its combination, we used a custom cNDA microarray to screen gene expression on ischemia mouse hippocampus. Cerebral ischemia was induced in anesthetized mice by urethane (1.5g/kg, i.p) to 38-48g male or female mice by occlusion of the middle cerebral artery (MCA) for 15 minutes twice with microvascular clips, followed by reperfusion for 10 minutes after the first occlusion. Experimental animal were divided into groups: Sham-operated, vehicle (M3,ischemic mice 3 hour;12 hour;24 hour),Baicalin(BA) (5mg/ml), Jasminoidin(JA) (25mg/ml),Jasminoidin(H) (50mg/ml),Cholic acid(CA) (7mg/ml),Concha margaritifera(CM) (50mg/ml), Nimodipine (NI) (50mg/ml),and JA+BA, JA+CA, JA+BA+CA, JA+BA+CA+CM. The prescription of combination therapies was composed of the same ratio on every compound. Treated compound dosage according to 0.2ml physic liquor/100g mouse or rat body weight treated at 1.5h post operation given through tail vein. Sham-operated mice underwent identical procedures except therapy were vehicle (2ml/kg; 100% DMSO). Infarct Volume and Neurological Score were tested to display variation on comeout change. Hippocampus (subfields CA1-4 of Ammon's horn) were carefully dissected out from re-anesthetized mice under RNAase-free conditions, flash frozen and stored at -75 C. Total RNA of each sample was extracted with Trizol reagent (Invitrogen, procedure according to the instruction manual). Three animals per group were pooled and homogenized in denaturing solution with a Polytron. Three pooled per group and nine times repeted microarray experiments of each pooled were preformed. Each microarray contained 374 cDNA derived from cDNA library of mouse brain (Invitrogen, Cat.1065-025). Plasmid magnified using Invitrogen provided library. Design and synthesize Primer according the information provided by selected genes.Total gene segment is 416, two of them is control(lambda-A and pUC19). The criterion of qualified PCR extending is: the total gained volume of the purpose strap DNA is greater or equal to 7?g;the purpose strap DNA gained volume/total DNA gained volume is greater or equal to 80%.Finally, the DNA concentration is adjusted to 0.5 ?g/ ?l.After refinded PCR_for every gene, 2?l DNA solution was taken to proceeding electrophoresis on the electrophoresis condition of 2%Gel.Then fix quantify analysis according electrophoresis photo have taken.Choosing 5 PCR refined products sequencing from two direction after amplification.These 5 genes all chosen from UniGene sample which marked by ?,one of them is used Dalian ? as cyclostyle to amplification.The five sequence genes was certified as aimed gene segment by Blast(Completed in TaKaRa(Dalian))

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Transcription profiling of mouse livers of adult and newborn animal response to Aflatoxin B1

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