Fat-1 transgenic cattle as a model to study the function of ω-3 fatty acids

Guo, Guo Tao, Liu, Ding, Yang, Nie, An, Guo Hong

Description

ω-3 polyunsaturated fatty acids have been shown to play an important role in health. Enriched with ω-3 polyunsaturated fatty acids modulate expression of a number of genes with such broad functions as cell proliferation, growth and apoptosis and cell signaling and transduction, these effects, seem to regulate coronary artery disease, hypertension, atherosclerosis, psychiatric disorders and various cancer. In this context, fat-1 transgenic cattle was designed to convert ω-6 to ω-3 fatty acids could form an ideal model to study the effect of ω-3 fatty acids on the above functions. This study focuses on the total genomic difference of gene expression between fat-1 transgenic cattle and wild-type using cDNA microarrays, several genes were found to be overexpressed or suppressed in transgenic cattle relative to wild-type, these discrepancy genes related with lipid metabolism, immunity, inflammation nervous development and fertility.

Sujets

• Gene expression

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 6
• Langue: English

Extrait

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RESEARCH

OpenAccess

Fat-1transgeniccattleasamodeltostudythe
functionof
ω
-3fattyacids
TaoGuo
1,2
,XinFLiu
1
,XiangBDing
1
,FeiFYang
1
,YongWNie
1
,YuJAn
2
andHongGuo
1*

Abstract
ω
-3polyunsaturatedfattyacidshavebeenshowntoplayanimportantroleinhealth.Enrichedwith
ω
-3
polyunsaturatedfattyacidsmodulateexpressionofanumberofgeneswithsuchbroadfunctionsascell
proliferation,growthandapoptosisandcellsignalingandtransduction,theseeffects,seemtoregulatecoronary
arterydisease,hypertension,atherosclerosis,psychiatricdisordersandvariouscancer.Inthiscontext,fat-1
transgeniccattlewasdesignedtoconvert
ω
-6to
ω
-3fattyacidscouldformanidealmodeltostudytheeffectof
ω
-3fattyacidsontheabovefunctions.Thisstudyfocusesonthetotalgenomicdifferenceofgeneexpression
betweenfat-1transgeniccattleandwild-typeusingcDNAmicroarrays,severalgeneswerefoundtobe
overexpressedorsuppressedintransgeniccattlerelativetowild-type,thesediscrepancygenesrelatedwithlipid
metabolism,immunity,inflammationnervousdevelopmentandfertility.
Keywords:
fat-1
,transgeniccattle,
ω
-3fattyacids,geneexpression,genefunction

Introduction
levelsof
ω
-3fattyacids,indicatingthatthetransgeneis
ω
-3fattyacidscanexertawiderangeofeffectsoncelltransmittable[2].
ω
-3fattyacidshavemanyimportant
function.Inadditiontobeingasourceofenergy,theseactionsnotonlybythemselvesbutalsobygivingraise
fattyacidscanactasdeterminantsofthephysiochem-tovariousbiologicallyactivecompounds.
ω
-3fattyacids
icalpropertiesofcellmembranes,assubstratesfortheplayasignificantroleinvariousdiseasesandespecially
productionofsignalingmoleculesorfunctioningmedia-incancersandneurological/psychiatricdisorders[2-5].
tors,andasmodulatorsintheregulationofgeneexpres-Duetothepolyunsaturatedfattyacidsmodulatedgene
sion.Therefore,
ω
-3fattyacidscanprofoundlyaffectthetranscription.Consideringthis,weutilizethecDNA
physiologicalactivityandpathologicalprocessthroughmicroarraythatisapowerfulmethodthatallowsthe
differentmechanisms.expressionofthousandsofgenestobedeterminedsimul-
Mammalscannotconvert
ω
-6to
ω
-3fattyacidsauto-taneously.Thestudiesofgeneexpressionwereregulated
matically.Fat-1transgenicmiceshowedthatincreasedby
ω
-3fattyacidsmostlyonspecifictissue
invitro
or
vivo
contentof
ω
-3fattyacids,especiallyALA,EPA,DHA,[2,6],therearerarereportsthegenomicexpressioninflu-
inaddition,theratioof
ω
-6/
ω
-3fattyacidsisdramati-encedby
ω
-3fattyacids,specificallyinfat-1transgenic
callydecreasedinvariouskindsoftissues[1].Fat-1cattle.Herewetakethefat-1transgeniccattleasmodelto
transgenicanimalmodeloffersanopportunityforinves-studythechangeofgenomicexpressioninfluencedbythe
tigatingthebiologicalfunctionsof
ω
-3fattyacidsandincreased
ω
-3fattyacidsanddecreasedratioof
ω
-6/
ω
-3
theimportanceoftheratioof
ω
-6/
ω
-3invariousphy-fattyacidsinthebody.Thousandsofdiscrepancygenes
siologicalprocessesanddiseases.Thetransgenicmicegeneratedfromthisexperiment,wechoosetherepresenta-
wasfoundtobenormalandhealthyandmanygenera-tivedatestoanalysisanddelineatetheexactmolecular
tionsoftransgenicmouselineshavebeenexaminedandmechanismoffunctionsof
ω
-3fattyacids.
theirtissuefattyacidprofilesshowedconsistentlyhigh
Materialsandmethod
Fat-1transgeniccattle
*Correspondence:[e-mail]
1
DepartmentofAnimalScience,TianjinAgricultureUniversity,Tianjin300384,
Cattlewereengineeredtocarry
fat-1
genefrom
Caenor-
China
habditiselegan
swhichcanaddadoublebondintoan
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Guoetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.

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unsaturatedfattyacidhydrocarbonchainandconvert
ω
-
6to
ω
-3fattyacids.Thetransgeniccattlewereprovided
byInnerMongoliaUniversity,lifescienceinstitute.
RNAisolationandanalysis
RNAwasextractedfromwholebloodbyTRIzolextrac-
tionprotocol.Toensurethequality,totalRNAwas
quantifiedbyUVspectrophotometry,andthepurityof
totalRNAwasassessedby1%agarose.
PurificationofRNAandcDNAsynthesis
IfthepurityoftotalRNAwasnotverywell,itwillbe
influencetheefficiencyofprobelabelingandtheresult
ofthechiphybridization.RNAwaspurifiedbyusinga
RNeasy
®
MiniKit(QIAGEN,Germany),followingthe
manufacturer
’
srecommendedprotocol.
One-stepofcDNAsynthesis.Thereactionwereper-
formedwith11.5ulofRNAmixture(2ugofpurified
RNA,5ulofT7promotorprimer,RNase-freeWater
addto11.5ul,thenincubationfor10minat65°C,ice-
bathfor5mintodenaturation),4ulof5×Firststrand
buffer,2ulof0.1MDTT,1ulof10mMdNTPmix,1
ulofMMLVRT,0.5ulofRNaseout.Thereactioncon-
ditionwasusedlidtemperatureat65°C,incubationfor
2hat40°C,65°Cfor15min,4°Cfor5min.
cRNAsynthesislabelingwithaaUTPandpurificationof
NRcAFirst,transcriptionmixture(60ul)including5.7ulof
RNase-freewater,20ulof4×Transcriptionbuffer,16
ulofNTP(10mM),6ulof0.1MDTT,6.4ulof50%
PEG,4ulofaa-UTP(25mM),0.5ulofRNaseOUT,0.6
ulofInorganicPyrophosphatase,0.8ulofT7RNA
Polymerase.Afterward,20ulofcDNAwasaddedinto
60uloftranscriptionmixandmixing.Thereaction
conditionwasusedlidtemperatureat60°C,incubation
for2hat40°C.
cRNAwaspurifiedbyusingaRNeasy
®
MiniKit(QIA-
GEN,Germany),followingthemanufacturer
’
srecom-
mendedprotocol.
Fluorescencelabelingandpurification
Toconcentratethe4ugofcRNAwhichwasabove
-mentionedto6.6ulandadd10ulofDMSO,3.4ulof
0.3MNaHCO3(pH9.0)andmixing.Cy3wasaddedinto
the20ulofmixture,incubationfor1hat25°C.Finally,
10ulof4MHydroxylaminewasaddedandincubation
for15minat25°C.FluorescencelabelingcRNAalso
needpurification,themethodassameasthepurifica-
tionofcRNA,whichwasabove-mentioned.
Hybridization(4×44Kmicroarrays)
ThepurifiedCy3cRNAdemandtofragmentation
beforethehybridization,thereaction(55ul)was

Page2of10

performedwith875ngofCy3cRNA,11ulof10×
BlockingAgent,2.2ulof25×FragmentationBuffer,
Nuclease-freewateraddedto55ul,incubationfor30
minat60°Ctofragmentation.45ulof2×GExHybridi-
zationBufferwasaddedintothecRNAfragmentation.
100ulmixturewasdroppedontothecenterofthearray
surfaceandthencoveredwithacoverslipwithoutany
bubbles.Theslideswereplacedintoasealedcassetteto
hybridizeat65°Cwaterbathfor17h.
Afterhybridization,themicroarrayslideswerewashed
oncewith2×SSC,0.1%sodiumdodecylsulfate(SDS)
at42°Cfor4min,oncewith0.1×SSC,0.1%SDSat
roomtemperaturefor10minandthreetimeswith0.1
×SSCatroomtemperaturefor1min.Themicroarray
slideswerethenwashedwithdistilledwaterandspin
dried.Hybridizedslideswerescannedat5
μ
musingan
AgilentchipScanner.Thescannercouldscanwith
100%and10%PMTautomatically,tworesultswere
combineduseAgilentsoftwareautomatically.
Resultandanalysis
Fat-1transgeniccattleandwild-typecattlehave43653
discrepancyexpressedtranscriptsaccordingtotheAgi-
lentsoftware.Itwillbewasteabundanttimeandenergy
toanalysisalldatabase,andsomedatabasesaremean-
inglesstoanalysis,sothisstudywechoosedifferentially
expressedgenesofp-value
≤
0.05andfc
≥
1(Table1).
Inourstudyfat-1transgeniccattleconvert
ω
-6fatty
acidsinto
ω
-3fattyacidsanddecreasetheratioof
ω
-6/
ω
-3fattyacids(datesnotshown),thechangecomposi-
tionofpolyunsaturatedfattyacidscaneffectsongene
expression,somegenesareupregulationandsome
genesaredownregulation,andthenaffectthephysiolo-
gicalactivityandpathologicalprocessthroughdifferent
mechanisms.
ω
-3fattyacidsonlipidmetabolism
Fat-1transgeniccattleenriched
ω
-3fattyacids,
ω
-3fatty
acidsplayamajorroleintheregulationofseveralgenes
involvedinfattyacidmetabolism.Therehadbeen
reportedthatthein
fl
uencedby
ω
-3fattyacidsonlipoly-
ticandlipogenicgeneexpression[7-9].Hyperlipidemia
isoftenassociatedwithinsulinresistance,coronary
arterydisease,hypertension[3