DOI:.38/n2816 Primr pir C lox P lox P Primr pir B Primr pir A - WT CM KO WT ES -50 kd -37 kd flox/flox X ZP3 Cr/Cr flox/+ -ZP3 Cr/+ flox/flox -ZP3 Cr/+ flox/flox -ZP3 Cr/+ X X flox/flox flox/flox Ooyts without mtrnl -Flox -Wil-typ -Cr Gn xprssion rltiv to Hprt1 1-1 -2-3 -4 Ctr1 Singl ooyt Ctr2 Ctr3 Ctr4 KO1 KO2 KO3 KO4 Fgf4 Tx1 Utf1 Nnog Sox2 Sll4 Stll Brg1 T4 Cx2 Eoms Gt4 Hprt1 f g Littr siz of flox/flox -ZP3 Cr/+ fml mi 12 8 6 4 2 0 Fml Ml 1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28-1454 p -250 p Offspring of flox/flox ZP3 Cr/+ fml mi WT -Ctr Figur S1 Elimintion of mtrnl A i not show ny fft on th ooyt s vlopmntl omptn. () Shmti rprsnttion of th A trgting DNA onstrut n position of th gnotyping primr sts moifi from Khlr t l. (2004). Fill rrowhs: lox P sits; Ovl ox: promotr; fill rtngls: xons 1 5. () Mting strtgy to gnrt mtrnl A-null ooyts. Th flox/flox mi wr mt with ZP3 Cr/Cr mi to prou offspring with th flox/+ /ZP3 Cr/+ gnotyp. Thn th flox/+ /ZP3 Cr/+ ml mi wr kross with flox/ flox fml mi to otin flox/flox /ZP3 Cr/+ mi. Th ml mi of this gnotyp wr kross with flox/flox mi gin to otin flox/ flox /ZP3 Cr/+ fml mi tht woul prou A-null ooyts. () Th tils of offspring wr ut n gnotyp with primr pir B to tt for th prsn of th flox lll (449 p), Wt lll (415 p), n primr pir for Cr (373 p). Th lst ln ws us s ngtiv ontrol without ing ny DNA. () Vlition of limintion of mtrnl A t th protin lvl y Wstrn lot nlysis. Ooyt smpls ompris xtrts from 400 or mor GV ooyts. A monlonl A ntioy tt wk n of protin in wil-typ ooyts (WT) n vry strong n in th ES ll smpl (ES) of pproximtly 45 kd, ut not in th -knokout ooyts (KO) n umulus lls (CM). () Gn xprssion of singl ooyts t th GV stg, nlyz y Fluiigm qrt-pcr using th Biomrk 48.48 Dynmi Arry systm (Fluiigm) furthr onfirm th limintion of th A trnsript without signifint impt on th xprssion of ooytn ling-spifi gns xmin. Th numr (1, 2, 3, or 4) right ftr th rvition (Ctr, for wil-typ ontrol, or KO, for knokout) rfrs to th iologil rplits. (f) A-knokout ooyts n support stlishmnt of totipotny, whih is nssry for full-trm vlopmnt, s shown y th norml littr siz from th rossing of flox/flox /ZP3 Cr/+ fml mi with CD1 wil-typ ml mi. (g) PCR gnotyping of th offspring from th ov rossing onfirm tht th A lll h n lt. Th rs rprsnt th mns from 3 thnil rplits, rsult rprsnttiv of h iologil rplit in. Th unropp vrsion of is shown in Supplmntry Fig. 5 n sour t for r shown in Supplmntry Tl 5. WWW.NATURE.COM/NATURECELLBIOLOGY 1
WT- - Gn xprssion rltiv to Control 1-1 -2 Iniviul lstomrs from iopsy Iniviul 8-ll mryos -Ctr -362 p -245 p Nnog Sox2 Fgf4 Utf1 Cx2 Eoms Tx1 Tp Ctr KO M-Z KO Mtrnl KO Zygoti KO WT M-Z KO Control Nnog Mrg Trom-1 Mrg Numrs of positiv lls 120 0 80 60 40 20 0 E4.5 mryos Ctr KO 89.00 60. 8.38 2.49 7 2.54 Nnog Nnog+Cx2 Cx2 Figur S2 Phnotyp of A-null mryos () Gnotyping with nst PCR on singl iopsi lstomrs from mryos otin y rossing flox/flox /ZP3 Cr/+ fml mi with +/- ml mi. () Quntittiv RT-PCR on singl gnotyp 8-ll mryos with triplits shows tht limintion os not ly tivtion of Nnog gn trnsription. Ctr: A +/, KO: mtrnl n zygoti Aknokout. () Immunoytohmistry of E2.5 mryos for Nnog (grn) n (r), M-Z KO: mtrnl n zygoti A knokout. () Immunoytohmistry of E3.5 lstoysts for Trom-1 (r), nothr TE mrkr, n (grn) loliz th protin to th TE, whih furthr onfirm th ling sprtion of ICM/TE in A-null mryos. () Avrg ll numrs of Nnog- n Cx2-positiv lls pr E4.5 mryo wr ount on onfol immgs of immnostin mryos. KO: A-knokout; M-Z KO: mtrnl n zygoti A knokout. Th sl rs rprsnt 25 mm in n. Vlu rprsnts mn±s.d. of 3 iologil rplits in n mn±s.d. of 61 n 41 mryo smpls for wiltyp n A KO, rsptivly in. Sour t for r shown in Supplmntry Tl 5. 2 WWW.NATURE.COM/NATURECELLBIOLOGY
Flox- WT- - -379 p -345 p -245 p 1 2 3 4 5 6 7 8 9 11 12 13 14 -Ctr Fluorsn intnsity of -GFP 60 E2.5 n=8 50 n=16 40 30 n=12 n=12 20 Ngtiv Control sigfp sisll4 160 140 120 0 80 60 40 20 E3.5 n= n=8 n=12 n=13 0 Ngtiv Control sigfp sisll4 Exprssion rltiv to E3.5 Blstoysts Tpt1 sitpt1 1.6 1.4 E3.5 Blstoysts Zsn4 sizsn4 Exprssion rltiv to E3.5 Blstoysts Esrr siesrr E3.5 Blstoysts Utf1 siutf1 Figur S3 -GFP xprssion is tivt in A-null mryos. () At th n of th tim-lps osrvtion on -GFP xprssion, h iniviul mryo ws mrk y numr with its gnotyp (mtrnl/zygoti) s trmin y. () Gnrt y rossing flox/flox /ZP3 Cr/+ fml mi with OG2-GFP +/- - +/ ml mi, GFP-xprssing E4.5 mryos wr slt s shown. Gnotyping of ths mryos rvl tht hlf (17/36) wr mtrnl/zygoti knokout n suggst tht OG2-GFP ws still tivt in A-null mryos. () Gnotyp ws trmin y nst PCR with orrsponing numrs to. () -GFP xprssion ws not fft in E2.5 (lft) n E3.5 (right) mryos following injtion of sisll4 into zygots otin y rossing flox/flox /ZP3 Cr/+ fml mi with GOF18-GFP ml mi. Emryos without th -GFP trnsgn wr us s ngtiv ontrol n sirna trgting GFP (sigfp) ws us s positiv ontrol. Fluorsn intnsity ws quntifi y ImgJ softwr. () Effiint knokown of Tpt1, Zsn4, Esrr n Utf1 t th mrna lvls y injtion of sirna uplxs s ssss y rl-tim RT-PCR. No signifint fft on xprssion ws osrv. Exprssion lvls of Hprt1 wr us s th intrnl ontrol to normliz th t n sigfp-injt mryos wr us s lirtors. Sl rs rprsnt 50 mm in n. Th rror rs rprsnt mn±s.e.m. of 8-16 iologil rplits in n mn±s.d. of 3 iologil rplits in. Th unropp vrsion of is shown in Supplmntry Fig. 5 n sour t for r shown in Supplmntry Tl 5. WWW.NATURE.COM/NATURECELLBIOLOGY 3
OtA4KO ooyt NT WT ooyt NT AKO ooyt PA WT ooyt PA Exprssion lvl rltiv to ESCs Nnog Mrg Gn xprssion rltiv to ES 3 2 1-1 -2-3 NT Blstoysts Fgf4 Utf1 Nnog Sox2 Sll4 Stll Dzl Cx2Eoms Gt4 ES Ctr1 Ctr2 Ctr3 KO1 KO2 KO3 PA1 PA2 PA3 DIC CAG-mRFP -GFP * * Figur S4 A-null ooyts rprogrmm somti ll nuli to pluripotnt sttus. () Immunoytohmistry of E4.0-NT lstoysts show tivtion of Nnog n xprssion y A-knokout (A KO) ooyts. WT: wil typ; NT: Nulr trnsfrr; PA: prthnogni. () Gn xprssion profiling of NT lstoysts rvl tivtion of xprssion of th pluripotnt gns n Nnog without mtrnl A xprssion. Th gn xprssion lvls wr otin with pools of 3 lstoysts with triplits n prsnt in omprison with ES lls (ES). Ctr: NT mryos using wil-typ ooyts; KO: NT mryos using A-knokout ooyts; PA: prthnogni mryos using -knokout ooyts. Th numr (1, 2, or 3) right ftr th rvition (Ctr n KO) rfrs to th iologil rplits. () Morphology of NT-ES lls grown on MEFs xprssing CAG-mRFP n -GFP. () Histology of trtom from NT-ES ll lin RG6 4 wks ftr injtion into SCID mi s ssss y hmtoxylin n osin stining. Th trtom ontin lls of ll 3 mryoni grm lyrs. Uppr lft pnl: krtiniz strtifi squmous pithlil lls (torml); uppr right pnl: nurl rostts (torml); lowr lft pnl: strit musl (msorml); lowr right: ilit olumnr pithlil lls jnt to pnrti inr lls (oth norml). () A littr of nontl NT-ES ll riv pups livr y srn stion on E19.0. In this prtiulr littr, 8 pups show norml full-trm vlopmnt, of whih on ws n on fil to initit rthing (*). Th sl rs rprsnt 30 mm in, 0 mm in n 50 mm in. Vlus rprsnt mn±s.d. of 3 iologil rplits. Sour t for r shown in Supplmntry Tl 5. 4 WWW.NATURE.COM/NATURECELLBIOLOGY
Figur S5 Unropp figurs for Fig. 1, 2, 2, 4 n Supplmntry Fig. 1 n 3. WWW.NATURE.COM/NATURECELLBIOLOGY 5
Supplmntry Tl lgns Supplmntry Tl 1 Squning rsults of RT-PCR mplion using primrs spnning xon 2 n xon 3 of th gn in A-null ooyts n mryos. Supplmntry Tl 2 Squning rsults of RT-PCR mplion using primrs spnning xon 3 n xon 4 of gn in A-null ooyts n mryos. Supplmntry Tl 3 Primrs for gn xprssion stuy. Supplmntry Tl 4 sirna trgt squns Supplmntry Tl 5 Sour t fil. This fil inlus th originl sour t for rl-tim RT-PCR ssy. Lgns to Supplmntry Vios Supplmntry Vio 1 Tim-lps roring of in vitro vlopmnt of A-null 8-ll mryo A iopsi n gnotyp morul with mtrnl/zygoti A-null ws ultur on MEFs in ES ll mium n osrv on th stg of mirosop with n inution hmr (TOKAI HIT, Jpn) fill with 5% CO 2 in ir n mintin t 37 C. Brightfil piturs wr tkn vry 5 min for 4 ys n wr ompil into movi with 24 frms pr son. Th vio monstrts tht A-null mryos initit vittion n form grossly norml-looking lstoysts with istint ICM. Howvr, immunostining of th outgrowth (Fig. 3) show ytoplsmi loliztion of Nnog s wll s frgmnttion of nuli. Supplmntry Vio 2 Tim-lps onfol roring rvl tivtion of -GFP xprssion in mtrnl-knokout n mtrnl/zygoti-knokout mryos Twlv 2-ll mryos from th mting of flox/flox /ZP3 Cr/+ fml mi with A +/ /-GFP +/+ ml mi n 4 mryos (#1, 3, 4 n 8) from th mting of flox/flox fml mi with A +/ /-GFP +/+ ml mi wr pl in KSOM AA in glss ottom ish with th sm onition s Supplmntry Vio 1 for onfol xmintion with 488 nm lsr. A onfol pitur h n tkn vry min for 3 ys n ws ompil into movi with 24 frms pr son. Th vio monstrt tht rgrlss of th gnotyp, ll mryos tivt -GFP t roun E2.5 in timly fshion, s i wil-typ mryos. Th gnotyp of h mryo is shown in Fig. S3. Supplmntry Vio 3 Brightfil tim-lps roring of th sm mryos t th sm tim point s Supplmntry Vio 2 This vio ws us to monitor th vlopmntl stg of th mryos n to tr th position of iniviul mryos for gnotyp trmintion. 6 WWW.NATURE.COM/NATURECELLBIOLOGY