RogerH.Reeves,Ph.D.

Dept.ofPhysiology
McKusickNathansInstituteforGeneticMedicine
JohnsHopkinsUniversitySchoolofMedicine
rreeves@jhmi.edu
Dr. Reeves research is supported by NICHD under PHS award 5R01-HD038384; The LuMind
Foundation (formerly Down Syndrome Research and Treatment Foundation (DSRTF) and
Research Down Syndrome (RDS); by The Anna and J ohn Sie Foundation and Global Down
syndrome. He is a consultant for Transcept Pharmaceuticals.
He serves on Science Advisory Boards of the LuMind Foundation; Research Down
Syndrome; the Linda Crnic Institute; the LonDowns Project and PHENOMIN, a French
incarnation of the IMPC; and is on the SciCAB of the National Down Syndrome Society where
he is also a member of the Board of Directors; none of these positions are remunerated.
Reproducibilityofexperimentswithmice:
Whatstheproblemandwillnewmouse
technologiessaveus?
Reproducibility:Whatstheproblemandwill
newmousetechnologies saveus?
1. Newtechnologies willnotsaveusbecausetherootproblemisnot
technology
Somepeoplearebetteratsomethingsthanothers
Cuttingedgescienceisnearlyalwaysdonebypeoplewhoaretechnicallybetterand
arebettercriticalthinkersthanthosewhocomealonglatertosayitisntright
2.Thereisafundamentalmisunderstandingofwhattheliteratureis,bythe
public,bythepressand,toaworryingdegree,byscientists
Inpublicperception,theScientificLiterature=InfallibleTruth.Anydeviationfrom
absolutetruthmustbetheproductoffraud,deceitorincompetence.
Theliterature ISSUPPOSEDTOBEWRONG thestockmarketofscientificideas.
3. Thereislittlerewardandmuchcostforformingahypothesis, doingeverything
youcanthinkoftodisproveit,andpublishingitwhenyoucantdisproveitso
thatalargergroupofmindscantrytodisproveit.
4. Thereissubstantialreward andenormouspressuretogetapaper.
MouseasaGeneticModelSystemfor
MammalianGenetics
1.Geneticstructuresinmicearenotfoundinnature
Implications ofinbredmice
2. MakeyourownGenotype
3. MakeyourownPhenotype
4. Comparativegenetics
MouseasaGeneticModelSystemfor
MammalianGenetics
Inbreeding:Genetichomogeneity doesnotmeanphenotypic
homgeneity.
(B6/N isnotthesamemouseasB6/J)
(Keeptrackofthemitochondriallineages)
Thereasonthatmammalsareoutbredistomaintaina
sufficientlydeepgenepoolofvariationsothatthespecies
canpersistinthefaceofeffectivelyinfiniteenvironmental
variation.
MouseasaGeneticModelSystemfor
MammalianGenetics
Recommendation 1:
Justify theuseofinbredvs.outbredmicewhenmodeling
humanconditions.Humanbeingsareoutbred.
MammGenome.2008June;19(6):382389.
CollaborativeCross
Makeyourowngenotype
1. Transgenicmice(pronuclearinjection)HoganB,Beddington R,
Constantini F,LacyE.ManipulatingtheMouseembryo:Alaboratorymanual.1994.
ColdSpringHarborLaboratoryPress.
2.Knockout(nullalleles)and Knockinmice(mutations,reporters),
tissuetargetedandconditionalmutations
ShinMK,Levorse JM,IngramRS,Tilghman SM.Thetemporalrequire
ment forendothelin receptorBsignalling duringneuralcrestdevelopment.
Nature.1999Dec2;402(6761):496501.
[THENEWSYNTHESIS GENOMEEDITING]
3.Chromosomeengineering
RamirezSolisR,LiuP,BradleyA.Chromosomeengineeringinmice.
Nature.1995378(6558):7204.
4.Geneediting
Mali,P.,Yang,L.,Esvelt,K.M.,Aach,J.,Guell,M.,DiCarlo,J.E.,Norville,J.E.,
andChurch,G.M.(2013).RNAguidedhumangenomeengineeringviaCas9.
Science 339,823826.
Makeyourowngenotype
Firststop,MGI!
http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=markerQF
HasKOMPalreadymadeyourmouse?
36,606genesintheIMPCdatabase
assignedfortargeting
EScellsavailable
micemade
mice phenotyped
MicroinjectionofDNAintoamalepronucleustocreateatransgenicmouse.
Upregulation
Dominantnegative
Gainoffunction
Complementationacross
species
http://www.med.ic.ac.uk/db/dbbm/tgunit.htm
Preimplantation mouseembryos.Hoganetal.,Manipulatingthemouseembryo,1994.
A26ganeedleisusedtointroduce
injectedzygotesintotheuterusof
apseudopregnantrecipient.Pups
arebornca.18dayslater.
Transgenicmice(pronuclearinjections)
canbeusedto
1. Modeldominantgeneticconditions
2. Creategeneticallymarkedpopulationsofcells
3. Surveygenomeregions(crypticfunctions)
4. Promoterbashing
5. CRISPR geneeditinginvitro
Embryonicstemcellsand
inducedPluripotentStemcells
Nature458,962965(2009)
Embryonicstemcellscanbemanipulated
inculture,theninjectedintomouseblasto
cyststoformpartoftheinnercellmass
(ICM).TheICMwillformtheembryo
properwhilethesurroundingtrophecto
dermcellscontributetoextraembryonic
structures(placenta).
UsingEScells:
Blastocystinjection
ChimerasformedfromEScellsandhostcells.
Achimerashasfourparents,butindividualcellshavegeneticinformationfromonly
oneofthetwopairs.
Embryonicstemcellderivedmice
canbeusedto
1. Modelrecessivegeneticconditions(knockin)
2. Creategeneticallymarkedpopulationsofcells
3. Findexpressedgenes(Promoter/enhancertrap)
4. Expressgenesconditionally(specifictimes
andplaces)
5.Mutationlibrary
Genomeediting
ZNFs(Zincfingernucleases)
TALENs(Transcriptionactivatorlikeeffector nucleases
CRISPR(ClusteredRegularlyInterspacedShortPalindromic Repeats)
Genecopia
Genomeediting
CRISPR(ClusteredRegularlyInterspacedShortPalindromic Repeats)
Science339,823(2013);
Genomeediting
AdvantagesofGenomeediting
1. Dogeneticengineeringinvivo nocellculturebasedsteprequired
2. Usestemcells(ESoriPS)ornot ifyouusethem,CRISPR greatly
increased efficiency
3. CanbeusedinanythingthathasDNAandisalive(bacteria;plants;
worms,flies,invertebrates,vertebrates)
4. Mice:Sometimestargetbothallelesinonegeneration
5. Mice:Targetmultiplelociinonegeneration
Disadvantages
1. Offtargetcutting(computeralgorithmsminimizeandpredictsites)
2. Formouse:Testingvectorsforproperregulation/responseincultured
cellsbeforeinvestinginmakingananimalmodelcanbeuseful,
especiallywherecomplicatedregulationorspecificsequencevariationis
required
TimelinesforGenomeEditing
EScelltargeting
1. Createconstruct(13months)
2. TargetEScells(5months)
a.Electroporate
b.Select100/expand/DNA
c.Screen
d.Expandpositivesubclones
e.Rescreen(Southern?)
f.ExpandforBCinjection
3. BlastocystInjectionhigh
percentagemalechimera
(2rounds,4months)
4. Germlinetransmission(25months)
[MayneedtorepeatBCinjectionhere]
[Mayneedtorepeatelectroporation]
5. Breedgermlinefounder(ifyouused
hybridEScellsthenyoumightwantto
inbreed addoneyearfor56
generations)
6. (Expandcolonyforexperiments)
Oneyearifeverythinggoeswell
Construct+$3500+$4850+salaryfor1year(0.3FTE)
CRISPR
1. Createconstructs(1month)
2. Pronuclearinjection(4wks)
3. Screen(PCR+sequence)(1wk)
4. (Expandcolonyeliminating
offsitetargets)
2months
$5000+0.05FTE
Notalltestswiththesamenamearethesametest
Recommendation 2:
Createminimumstandard phenotypingtests.
Behavior
Pharmacology
Metabolism
Phenotyping
1. Whoshouldsetstandards?
2. Howstandardizedshouldthisbe?
3. Howextensive/intrusive/directive?
Crabbe,J.,Wahlsten,D.,andDudek,B.(1999).Geneticsofmousebehavior:
interactionswithlaboratoryenvironment.Science 284,16701672.