Escolar Documentos
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ParadigmintheEraofNextGeneration
Sequencing
CraigFranklin,DVM,PhD,DACLAM
MMRRC,RRRC,MUMC
UniversityofMissouri
Archivesanddistributionrepositoriesofgenetically
engineeredmouseandratmodelsofhumandisease
OVERARCHINGQUESTION:Howaremodelsdistributed
influencedbydifferinggutmicrobiota(GM)
Complex gnotobiology
Gnotobiosis = known life
With next generation sequencing, we can better define
microbial OTUs present in a variety of sites
Complexcommunities
Validation
Observationgenerating
Complement classical
gnotobiotic studies
Definedcommunities
Questions
DocomplexGMsvaryincontemporaryrodent
colonies?
Whatfactorscause(correlatewith)suchvariation?
Doesvariationinfluencemodelphenotypes?
HowcanweexploitcomplexGMvariabilityin
animalmodeling?
Refinemodels?
Assessreproducibility?
Improvetranslatability?
DoesGMvary
in
contemporary
rodent
colonies?
Bacterialfamily
PLoS One.2015Feb12;10(2):e0116704
Producer
PLoS One.2015Feb12;10(2):e0116704
Diet?Bedding?Housing?
Cecal microbiota
Cecal microbiota
VentilatedRack/Aspen
VentilatedRack/Paper
StaticMI/Aspen
StaticMI/Paper
Whatfactorscause(correlatewith)variation?
Host
Genetics
Age
Sex? ongoing
Producer
Manipulation
Antibiotics
Disease
Shipping?
Sampling
Location
Timeofday
Extractiontechnique
Environment
Cagetype
Bedding
Diet(acutechronic)
Housingdensity
Watertreatment?
Socialinteraction?
Enrichment? ongoing
Lightcycle?
Production
Naturalvs.caesarian
rederivationorcryorecovery
Many,Manymore
Doesvariationaltermodelphenotypes?
M1=GMCRL
M2=GMJAX
M3=GMTAC
Doesvariationaltermodelphenotypes?
C3H/HeJ IL10/
Differingmicrobiotaassociatedwith
differingcolonictumorcounts
Host/microbiota interaction
CanweexploitexistingGM
variability?
Options
Axenic/germfree
Monocolonized
Definedflora
Humanized
Complexmicrobiota
Barrier/specific
pathogenfree(SPF)
Conventional
Wildtype?
Richness&DiversityofGM
Complex
Microbiota
Targeted
Rederivation
(CMTR)
IL10KO,Smad3KO,andApcMinmice;ApcPIRCrat
OutbredcolonieswithdifferingmicrobiotaforCMTR
CD1
Embryos
Embryo
Transfer
Surrogate Dams
(Different Vendors)
Crl:CD1
GMTAC
GMJAX
GMCRL
C57BL/6J
C57BL/6NTAC
CD1
Generation 1
CD1
Generation 2-6
OutbredcolonieswithdifferingmicrobiotaforCMTR
0.5
0.4
0.3
CD1GMCRL
CD1GMJAX
0.2
First generation
0.1
0
Second generation
-0.1
-0.2
CD1 GMTAC
-0.3
-0.4
-0.5
-0.8
-0.6
-0.4
-0.2
0.2
0.4
0.6
ComparisonofCMTRsurrogatesource
Originalsurrogates(CD1,B6,B6)
B6IL10/
CD1surrogates
Canweusebankedfecesto
reconstituteGM?
LowdiversitydonorHighdiversityrecipient HighdiversitydonorLowdiversityrecipient
ReciprocalpostantibioticGMtransfers
Ericsson,etal.,Reciprocalfecalmicrobiotatransferbetweenmicewithlowandhighrichnessmicrobiotausingfreshor
frozenfecesorfreshcecal contents.FrontMicrobiol (acceptedpendingrevisions)
Reconstitution
isincomplete
Ericsson,etal.,Reciprocalfecalmicrobiotatransferbetweenmicewithlowandhighrichnessmicrobiotausingfreshor
frozenfecesorfreshcecal contents.FrontMicrobiol (acceptedpendingrevisions)
AremurineGMtranslatable?
Embracecomplexity
ImpactonAnimal
WelfareandEthics
Improvereproducibilitybydefining
GMandminimizingvariability
Improvestudydesign
Optimizemodels
Biomarkersfornonterminal
endpoints?
Ultimatelydecreaseanimal
numbers
Manyfuturechallenges
Reportingmicrobiota
Consistentmethods,databasesetc.
$$$
Standardize?
Bioinformatics
Translatability
Correlationvs.causation
Metabolomics/function
Unculturable agents
Genome+Metagenomesurveys
Viruses,fungi,protozoa,etc.
MetagenomicsGenomics
Acknowledgements
AaronEricsson
&MUMC
ElizabethBryda
&RRRC
MUDNACore
MUInformatics
ResearchCore
Facility
J.AmosLandgraf
&lab
Yuksel Agca &lab
CatherineHagan
&lab
J.WadeDavis
&lab
U42OD01091817
P40OD01106216
ComparativeMetagenomicsLaboratory
(akapoopgroup)
T32OD01112637
Questions?