Regulation of pluripotency in male germline stem cells by Dmrt1Supplementaldata.docx
- 文档编号:10821499
- 上传时间:2023-05-27
- 格式:DOCX
- 页数:24
- 大小:423.69KB
Regulation of pluripotency in male germline stem cells by Dmrt1Supplementaldata.docx
《Regulation of pluripotency in male germline stem cells by Dmrt1Supplementaldata.docx》由会员分享,可在线阅读,更多相关《Regulation of pluripotency in male germline stem cells by Dmrt1Supplementaldata.docx(24页珍藏版)》请在冰点文库上搜索。
RegulationofpluripotencyinmalegermlinestemcellsbyDmrt1Supplementaldata
Supplementalinformationfor
RegulationofpluripotencyinmalegermlinestemcellsbyDmrt1
SeijiTakashima,MichikoHirose,NarumiOgonuki,MikiEbisuya,KimikoInoue,MitoKanatsu-Shinohara,TakashiTanaka,EisukeNishida,AtsuoOgura,TakashiShinohara
Supplementalinformationforthispapercontainsmaterialsshownbelow.
SupplementalFigureS1-10
SupplementalTableS1-5
Supplementalexperimentalprocedures
Supplementalreferences
SupplementalFigureS1.ConfirmationofKDbyrealtimePCR.(A)RealtimePCRanalysisofDnmt1inp53KOGScells7daysaftertransfection(n=3).(B,C)RealtimePCRanalysisofp53(B)andDnmt1(C)inWTKOGScells7daysaftertransfection(n=3).pSicoRwasusedasacontrol.
SupplementalFigureS2.ExpressionofGCTcandidategeneOE/KDinGScells.(A)ConfirmationofKDbyrealtimePCR.p53KOGScellswereinfectedwiththeindicatedlentivirusandgeneexpressionwasanalyzedbyrealtimePCR7daysaftertransfection(n=3).WTGScellswereusedforBaxKD.pLKO.1EGFPwasusedasacontrol.(B)ConfirmationofOEbyRT-PCR.p53KOGScellswereinfectedwiththeindicatedlentivirusandgeneexpressionwasanalyzedbyRT-PCR7daysaftertransfectionusingtransgene-specific(cDNA-IRES2)primerpair.AlentivirusvectorcontainingtheappropriatecDNA-IRES2sequencewasusedasapositivecontrol.CyclinE1wasco-transfectedwithcyclinD2topromoteitstumorigenicity(Leeetal.2009).CSII-EF1α-IRES2-hKO1wasusedasacontrolforHras,whereasCSII-EF1α-IRES2-Venuswasusedasacontrolfortherestofthegenes.(C)ConfirmationofOEbyrealtimePCR.p53KOGScellswereinfectedwiththeindicatedlentivirusandgeneexpressionwasanalyzedbyrealtimePCR7daysaftertransfection(n=3).CSII-EF1α-IRES2-Venuswasusedasacontrol.moi,multiplicityofinfection.
SupplementalFigureS3.DerivationofDmrt1-mGScells.(A)Developmentofepiblast-likecoloniesduringreprogrammingthatoccurredinGScellsderivedfromatransgeniclinethatexpressesEGFPunderthecontrolofaNanogpromoter.(B)DerivationofDmrt1-mGScellsfromadultWTDBA/2GScells.Bar=200μm(A),100μm(B).
SupplementalFigureS4.CharacterizationofDnmt1-andDmrt1-mGS.(A)RT-PCRanalysis.(B)Flowcytometricanalysis.(C)Nanogimmunostaining.(D)BisulfitesequencinganalysisoftheDMRsofH19andIgf2r.Opencircles,unmethylatedCpG;closedcircles,methylatedCpG.(E)Designofthep53KDandDnmt1KDvectors.EGFPandtheshRNAoligoareflankedbyloxPsites(yellowtriangles),whichcanberemovedbycre-mediatedrecombination.5’LTR,5’longterminalrepeat;Psi,packagingsignal;cPPT,centralpolypurinetract;CMV,cytomegaloviruspromoter;WPRE,woodchuckhepatitisvirusposttranscriptionalregulatoryelement;3’LTR∆U3,3’LTRwithdeletionoftheenhancerandpromotersequencesintheU3region.(F)Dnmt1-mGScellsinducedfromROSAGScells.Cre-mediatedrecombinationwasconfirmedbyextinctionofEGFPfluorescence.(G)Sectionofteratomasundertheskin,showingtissuesofthreegermlayers.(H)RT-PCRanalysisofspermatogoniamarkerexpressioninDnmt1-andDmrt1-mGScells.(I)COBRAoftailDNAfromoffspringofDmrt-mGSchimera.PCRproductsweredigestedwiththeindicatedenzymes.Percentmethylationisshownbelowthegels.U,non-cleaved;C,cleaved.Bar=100μm(C,G),50μm(F).
SupplementalFigureS5.RealtimePCRanalysisofDmrt1targetgenesinWT-GScellsafterDmrt1KD(n=3).pLKO.1EGFPwasusedasacontrol.Allexperimentswerecarriedoutwithconcurrentp53KD.
SupplementalFigureS6.ApoptosisofGScellsbytransfection.(A,B)Suppressionofapoptosisbyp53deficiencyafterSox2(A)orOct4(B)OE.Foreachcelltype,atleast82cellswerecounted7daysaftertransfection(n=5).CSII-EF1α-IRES2-hKO1andCSII-EF1α-IRES2-VenuswereusedascontrolsforSox2andOct4OE,respectively.(C)Suppressionofapoptosisbyp53deficiencyafterOctKD.Foreachcelltype,atleast100cellswerecounted7daysaftertransfection(n=5).pLKO.1EGFPwasusedasacontrol.
SupplementalFigureS7.WesternblotanalysisofOct4andSox2expressioninp53KOGScells4daysafterSox2OE.CSII-EF1α-IRES2-hKO1wasusedasacontrol.
SupplementalFigureS8.RealtimePCRanalysisofOct4targetgeneexpressioninp53KOGScellsafterOct1OE(A),Oct1KD(B),Oct4OE(C)andOct4KD(D)14daysaftertransfection.CSII-EF1α-IRES2-VenusandpLKO.1EGFPwasusedascontrolsforOEandKDexperiments,respectively.ExpressionofDax1,Fgf4,Lefty1,Pml,andNanogwasnotdetectableinGScells.
SupplementalFigureS9.CharacterizationofSox2-andOct4-mGS.(A)RT-PCRanalysis.(B)Flowcytometricanalysis.(C)Nanogimmunostaining.(D)Invitrodifferentiation.(E)BisulfitesequencinganalysisoftheDMRsofH19andIgf2r.Opencircles,unmethylatedCpG;closedcircles,methylatedCpG.(F)DesignoftheSox2/Oct4removableexpressionvector.ThecDNAinsertisflankedbyloxPsites(yellowtriangles),whichcanberemovedbycre-mediatedrecombination.DeletionofthecDNAinsertinitiatesDsRedexpression.5’LTR,5’longterminalrepeat;Psi,packagingsignal;cPPT,centralpolypurinetract;CMV,cytomegaloviruspromoter;WPRE,woodchuckhepatitisvirusposttranscriptionalregulatoryelement;3’LTR∆U3,3’LTRwithdeletionoftheenhancerandpromotersequencesintheU3region.(G)Sox2-mGSandOct4-mGSinducedfromgreenGScells.Cellswerethensubjectedtocre-mediateddeletionoftheSox2/Oct4transgene.Cre-mediateddeletionwasconfirmedbyexpressionofDsRedfluorescence.Bar=100μm(C,D),50μm(G).
SupplementalFigureS10.Scatterplotcomparisonoftheglobalgeneexpressionprofiles.Blacklinesindicateboundariesof2-folddifferenceingeneexpression.
SupplementalTableS1.SummaryofmGScellinduction
GScell
Genetransduction
Multiplicityofinfection
No.ofwells
cultured
No.ofwellswithmGScells
Daycoloniesexamined
p53KO
Dnmt1KD
2
3
2
28
Dmrt1KD
2
3
3
Dnd1KD
4.7
3
1
PtenKD
0.9
3
0
Cdk4OE
3.4
3
0
CyclinD2OE
CyclinE1OE
9.5
0.97
3
0
AktOE
0.3
3
0
β-CateninOE
1.7
3
0
HRasOE
0.8
3
0
Bcl6bOE
4.9
3
0
Fgfr3OE
4.8
3
0
Lats2KD
1.7
3
0
Sprouty4OE
3.2
3
0
KitLOE
4.7
3
0
TertOE
0.7
3
0
WT
pSicoR
20
6
0
pLKO.1EGFP
2
4
0
p53KD
10
22
0
Dnmt1KD
2
4
0
Dmrt1KD
2
7
0
Dnmt1KD
p53KD
2
10
15
3
Dnmt1KD
p53KD
10
10
12
9
Dmrt1KD
p53KD
2
10
30
21
Dnd1KD
p53KD
10
10
7
0
Sox2OE
p53KD
10
10
22
7
21
UTFOE
p53KD
10
10
12
0
p19KD
p53KD
10
10
20
0
p18KD
p53KD
10
10
12
0
Oct4OE
p53KD
10
10
31
12
Oct1KD
p53KD
10
10
12
3
Oct4OE
Oct1KD
p53KD
10
10
10
15
8
Oct4OE
Oct1OE
p53KD
10
10
10
8
3
Oct6KD
p53KD
10
10
12
0
Cells(5105)wereculturedin6-wellplates.
SupplementalTableS2.ContributionofmGScellstoembryonicdevelopment
Donorcell
Blastocyst
No.ofembryostransferred
No.ofembryosimplanted(%)
No.ofpupsborn(%)
Chimera
Male
Female
Dnmt1-mGS
B6
ICR
153
30
38(24.8)
NA
1(0.0)
10(33.3)
NA
1/4(25.0)
NA
0(0)
Dmrt1-mGS
B6
214
38(17.8)
2(0.9)
1/1(100.0)
0(0)
Sox2-mGS
B6
42
14(33.3)
9(21.4)
3/5(60.0)
1/3(33.3)
ICR
72
20(27.8)
9(12.5)
1/3(33.3)
2/5(40.0)
Oct4-mGS
B6
52
25(48.1)
3(5.8)
NA
NA
ICR
54
NA
21(38.9)
8/12(66.7)
4/8(50.0)
NA,notapplicable.Cannibalizedpupsarenotincludedinchimeraanalysis.
SupplementalTableS3.pLKO.1lentivirusvectorsusedinKDexperiments
Gene
TRCIDNumber
Bax
TRCN0000009668,TRCN0000009669,TRCN0000009670,TRCN0000009672
Dmrt1
TRCN0000084388,TRCN0000084389,TRCN0000084390,TRCN0000084391,TRCN0000084392
Dnd1
TRCN0000247049,TRCN0000247050,TRCN0000247051,TRCN0000257069
EGFP
TRCLentiviralEGFPshRNAPositiveControl
Lats2
TRCN0000022705,TRCN0000022706,TRCN0000022707
Oct1
TRCN0000175063,TRCN0000175090,TRCN0000240638
Oct4
TRCN0000009611,TRCN0000009612,TRCN0000009613,TRCN0000009614,TRCN0000009615
Oct6
TRCN0000075418,TRCN0000075419,TRCN0000075420,TRCN0000075421,TRCN0000075422
p18
TRCN0000085033,TRCN0000085034,TRCN0000085037
Pten
TRCN0000028990,TRCN0000028992,TRCN0000028993
Sox2
TRCN0000085748,TRCN0000085749,TRCN0000085750,TRCN0000085751,TRCN0000085752
SupplementalTableS4.PrimersforPCRandbisulfitesequencing
Gene
Forwardprimer
Reverseprimer
Akt
TTCCTCTCAAGAACGATGG
CTCTGTCTTCATCAGCTGGC
Akt-IRES2
AGATGATCACCATCACGCC
CGACATTCAACAGACCTTGC
β-catenin
AGCAAGCTCATCATTCTGGC
TGAACAAGTCGCTGACTTGG
β-catenin-IRES2
TTCACTCTGGTGGATACGGC
CTTATTCCAAGCGGCTTCG
Baf155
GATGTGTGTGCTGTGATGAGG
GAGGTGATCGAAGATGCAGG
Bax
AGGATGATTGCTGACGTGG
TGATGGTTCTGATCAGCTCG
Bcl6b
CCCGGGCTCAAGAGACTTC
TTCCTGGGCGGTGGATTAGC
Bcl6b-IRES2
GCAGCAGTGAAGAAGGAACC
CTTCGGCCAGTAACGTTAGG
c-myc
TGCAGGACCTCACCGC
CTTCTTGCTCTTCTTCAGAGTCG
Cdk4
TGCAGTCTACATACGCAACACC
TCGTCTTCTGGAGGCAATCC
Cdk4-IRES2
TAAGCCAATCTCTGCCTTCC
CGACATTCAACAGACCTTGC
Cripto
GGTCTATCCTCCATGGCACC
CATCACGTGACCATCACAGC
CyclinD2
TTCATTGAGCACATCCTTCG
TTCATCATCCTGCTGAAGCC
CyclinD2-IRES2
ATGATTGCAACTGGAAGCG
CTTCGGCCAGTAACGTTAGG
CyclinE1-IRES2
CTCAGGTTATCAGTGGTGCG
CTTCGGCCAGTAACGTTAGG
Dax1
ATCTGGAAGCAGGGCAAGTA
TCCTGTACCGCAGCTATGTG
Dmrt1
ATGAAGACCTCAGAGAGCCG
CAAGCCAGAATCTTGACTGC
Dnd1
AGTTCAGTACGCACCGAGC
GTTGACACAGCAGTTGCAGG
Dnmt1
ACTGGAAGAGGTAACAGCGG
GCCTCAATGATAGCTCTCTGG
Eras
TCAGATCCGCCTACTGCC
TTACCAACACCACTTGCACC
Esrrb
ACCATTCAAGGCAACATCG
CACACCTTCCTTCAGCATCC
Etv5
AACTTGGTGCTTCATGCTCC
ACTTAGCACCAAGAGCCTGC
Fbxo15
AGAAGCGTTGCTCTTCCTCC
AATGCAGCTGCGTACTTCC
Fgf4
GAGGCGTGGTGAGCATCTT
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- Regulation of pluripotency in male germline stem cells by Dmrt1 Supplementaldata
链接地址:https://www.bingdoc.com/p-10821499.html