Characterization and genetic manipulation of primed stem cells into a functional naïve state with ESRRB

Table 1 Naive and primed mouse embryonic stem cells

Mouse strain of ESC line	Targeted locus	Germline transmission Pluripotent	No germline transmission Not pluripotent
129Sv	POMC	I (QKQR-1E)	II (QKQR-11B)
C57BL/6	IGFR1	III (IGFR1-152)	IV (IGFR1-R13)
C57BL/6	FGF13	V (FGF13-1)	VI (FGF13-15)

ESC: Embryonic stem cell.

Table 2 Primers used for quantificational real-time polymerase chain reaction to amplify and quantify expression of differentially expressed genes

Mouse	Gene identification	Fwd primer	Rev primer
Oct-4	NM_013633.2	CCCCATGTCCGCCCGCATAC	AGGCCCAGTCCAACCTGAGGTC
Sox-2	NM_011443.3	GAAGAACAGCCCGGACCGCGT	ATGAACGGCCGCTTCTCGGT
c-myc	NM_010849.4	ACCCGCTCAACGACAGCAGC	ACTAGGGGCTCAGGGCTGGC
KLF-4	NM_207209.2	TAGTGGCGCCCTACAGCGGT	TCGTGTGTGTTGGGCCGGTG
KLF-5	NM_009769.4	CACCGGATCTAGACATGCCC	ACGTCTGTGGAACAGCAGAG
Pax-6	NM_001244198.1	CACCAGACTCACCTGACACC	TCACTCCGCTGTGACTGTTC
BMP-7	NM_007557.3	CTGAGTAAAGGACAGGGGCG	CTGAGTAAAGGACAGGGGCG
ESRRB	NM_001159500.1	CTACGCCACTCAAGAAGCCA	TTGATGAAGGAGCCGCAACT
Nanog	NM_028016.1	GGCTGCCTCTCCTCGCCCTT	GTGCACACAGCTGGGCCTGA
ERAS	NM_181548.2	TGCCCCTCATCAGACTGCTA	CCAAGCCTCGTGACTTTCCT
ATRX	NM_009530.2	CTTGCTTTGTTTCCGTGGCTCT	CTTGTTTCCACTCATGGGCTC
RNF17	XM_006519107.1	CACCTAGTGGAGAGTGACCA	TCTAAATGCCTGTCAGGGGC

Table 3 Top differentially expressed genes

ID	Gene symbol (HUGO)	t	P value
1420106_at	Siah1a	3.397282751	0.019877842
1451158_at	Trip12	3.397517988	0.019872832
1425223_at	Birc3	3.399188537	0.019837294
1455579_at	Csng	3.407711727	0.019657099
1416670_at	Setdb1	3.40836782	0.019643305
1448406_at	Cri1	3.417799448	0.019446231
1420981_a_at	Lmo4	3.424023676	0.01931741
1417831_at	Smc1l1	3.429111876	0.019212822
1423271_at	Gjb2	3.440985605	0.01897126
1425329_a_at	Dia1	3.444249087	0.018905474
1438223_at	Grid2	3.447575597	0.018838686
1422666_at	Cblc	3.458197089	0.018627226
1434755_at	Coro2b	3.458912253	0.018613086
1422812_at	Cxcr6	3.459767614	0.01859619
1416515_at	Fscn1	3.460527429	0.018581195
1449371_at	Harsl	3.460876887	0.018574304
1415772_at	Ncl	3.461800178	0.018556109
1448389_at	Wdr5	3.473565946	0.018326028
1426389_at	Camk1d	3.474454082	0.018308793
1424840_at	Rbks	3.476474953	0.018269645
1425234_at	1700051I12Rik	3.477915552	0.018241796
1452638_s_at	Dnm1l	3.478750478	0.018225678
1430335_a_at	Pax3	3.480374948	0.018194365
1427854_x_at		3.482574812	0.018152058
1452402_at		3.483987577	0.018124947
1438070_at	Phf3	3.487165588	0.018064131
1454061_at	Thumpd3	3.488257585	0.018043287
1420053_at	Psmb1	3.488906078	0.018030922
1422546_at	Ilf3	3.508209824	0.017667244
1418909_at	Ermap	3.509477595	0.017643654
1424498_at	5730596K20Rik	3.515771328	0.017527076
1418065_at	Rag2	3.520156261	0.017446374
1425961_at	BC016548	3.520765643	0.017435193
1444953_at	8430423A01Rik	3.524330508	0.017369944
1418227_at	Orc2l	3.526672877	0.017327223
1419179_at	Txnl4	3.529589461	0.017274198
1423249_at	Nktr	3.532283103	0.01722539
1427554_at	Hel308	3.53288254	0.01721455
1427643_at	1200009O22Rik	3.543340398	0.01702668
1421869_at	Trim44	3.554867777	0.016822308
1427482_a_at	Car8	3.558911802	0.016751276
1432459_a_at	Rog	3.561412525	0.016707523
1438245_at	Nfib	3.565204959	0.01664142
1422956_at	D1Pas1	3.576076558	0.016453576
1422036_at	Strn	3.57762187	0.016427073
1453683_a_at	1200008O12Rik	3.585757456	0.016288346
1419253_at	Mthfd2	3.586846457	0.016269879
1420974_at	Setdb1	3.587477754	0.016259184
1431686_a_at	Gmfb	3.588593964	0.016240294
1430586_at	2700007P21Rik	3.588969301	0.016233948
1423553_at	Dnajb3	3.599839679	0.016051375
AFFX-18SRNAMur/X00686_5_at		3.612923955	0.015834729
1438748_at	2700078E11Rik	3.613005797	0.015833384
1425605_a_at	Lmbr1	3.614705726	0.015805487
1443589_at	Gpm6b	3.615856465	0.015786634
1425338_at	Plcb4	3.61646714	0.015776639
1428132_at	Cdc42se1	3.625113293	0.015635909
1450541_at	Pvt1	3.628326703	0.015583973
1456565_s_at	Map3k12	3.631282846	0.015536369
1419052_at	Ovol1	3.635287213	0.015472151
1451456_at		3.640820903	0.015383907
1452642_at	Tmem16f	3.646863541	0.015288205
1451021_a_at	Klf5	3.651556285	0.015214353
1421933_at	Cbx5	3.652925906	0.015192876
1419241_a_at	Aire	3.655287704	0.015155921
1456190_a_at	BC031140	3.659632397	0.015088211
1436162_at	C730048C13Rik	3.668343947	0.014953486
1420517_at	2310010I16Rik	3.669025757	0.01494300
1452408_at		3.669339752	0.014938174
1438215_at	Sfrs3	3.674062823	0.014865793
1417757_at	Unc13b	3.696140227	0.014532751
1417270_at	Wdr12	3.706962408	0.014372629
1424942_a_at	Myc	3.714859557	0.014257063
1422135_at	Zfp146	3.722089586	0.014152193
1425270_at	Kif1b	3.723748371	0.014128258
1428045_a_at	Elf2	3.731380905	0.014018724
1427101_at	Metrn	3.749492087	0.013762692
1419940_at	4930488L10Rik	3.761372517	0.01359766
1435626_a_at	Herpud1	3.76528356	0.01354383
1423437_at	Gsta3	3.765317741	0.013543361
1449416_at	Fzd4	3.775661238	0.01340218
1428160_at	Ndufab1	3.778355433	0.013365684
1417029_a_at	Trim2	3.781146538	0.013327996
1455808_at	4922502D21Rik	3.782747632	0.013306432
1430483_a_at	2310042N02Rik	3.791162199	0.013193759
1424083_at	Rod1	3.791959817	0.013183136
1460725_at	Xpa	3.792592186	0.01317472
1452318_a_at	Hspa1b	3.796896622	0.013117602
1421230_a_at	Msi2h	3.812474518	0.012913258
1418349_at	Hbegf	3.817881032	0.012843195
1440192_at	1810054D07Rik	3.854206413	0.012383645
1452243_at	Kcnj14	3.85496781	0.012374217
1451887_at	Lrba	3.857465589	0.012343347
1419900_at	Sin3a	3.88287621	0.012034288
1450193_at	Hcn1	3.891121372	0.011935929
1417905_at	Prlpf	3.896151709	0.011876375
1434987_at	Aldh2	3.902236841	0.011804791
1449118_at	Dbt	3.905570325	0.011765788
1420982_at	Rnpc2	3.917975152	0.011621945
1424755_at	Hip1	3.936108008	0.011415316
1446914_at	Eif2s2	3.944832819	0.011317406
1455540_at		3.954421033	0.011210923
1456319_at		3.960519659	0.011143796
1438741_at	Rbm13	3.971304363	0.011026221
1449597_at		3.9720719	0.011017908
1416934_at	Mtm1	3.975422707	0.010981701
1426196_at		3.981642563	0.010914857
1427765_a_at	Tcrb-V13	3.988067373	0.010846303
1419277_at	Usp48	3.995642623	0.010766111
1438700_at	Fnbp4	4.007934467	0.010637441
1451739_at	Klf5	4.009661813	0.010619502
1452415_at	Actn1	4.013468651	0.010580091
1421595_at	9630031F12Rik	4.026451959	0.010446942
1423093_at	Incenp	4.056991536	0.010141312
1449729_at		4.059318965	0.010118447
1448371_at	Mylpf	4.060423682	0.010107615
1432007_s_at	Ap2a2	4.061118175	0.010100812
1426375_s_at	BC019806	4.061705841	0.01009506
1421267_a_at	Cited2	4.067131851	0.010042127
AFFX-18SRNAMur/X00686_3_at		4.068244684	0.01003131
1424872_at	2310001H12Rik	4.072170362	0.009993261
1448606_at	Edg2	4.076478623	0.009951696
1420930_s_at	Catnal1	4.093968451	0.009784998
1438082_at	2310028N02Rik	4.096841999	0.00975792
1418569_at	2410043F08Rik	4.109867254	0.009636263
1417178_at	Semcap2	4.111817999	0.009618195
1452620_at	Pck2	4.113798961	0.009599886
1419116_at	5430428G01Rik	4.127933982	0.009470413
1451902_at	BC021442	4.164797416	0.009142157
AFFX-18SRNAMur/X00686_M_at		4.172558479	0.009074738
1449838_at	Crisp3	4.181403551	0.008998604
1450430_at	Mrc1	4.194900667	0.008883849
1431893_a_at	Tprt	4.199779855	0.008842784
1453360_a_at	Tex9	4.212874201	0.008733659
1418417_at	Msc	4.222435464	0.008654964
1456225_x_at	Trib3	4.225132709	0.008632913
1427649_at	Wdr22	4.23098011	0.008585331
1452502_at	Serf1	4.234174345	0.008559468
1452070_at	Dedd2	4.27019116	0.008274009
1448457_at	Krt2-6g	4.278133662	0.008212555
1416268_at	Ets2	4.278591968	0.008209025
1425831_at	Zfp101	4.280752877	0.008192405
1449693_at	Map3k7	4.284119202	0.008166592
1449229_a_at	Cdkl2	4.286647093	0.008147271
1452142_at	Slc6a1	4.305051867	0.008008185
1430634_a_at	Pfkp	4.316779598	0.007920996
1428060_at	Cd3z	4.323698776	0.007870073
1434326_x_at	Coro2b	4.329579399	0.007827092
1424843_a_at	Gas5	4.357574401	0.007626183
1421279_at	Lamc2	4.404512781	0.007302618
1421496_at	2410116I05Rik	4.40646557	0.007289506
1419921_s_at	Usp7	4.409499533	0.007269189
1448348_at	Gpiap1	4.411257474	0.007257448
1423809_at	Tcf19	4.516818863	0.006591172
1429427_s_at	Tcf7l2	4.555365814	0.006365827
1431117_x_at	1810029B16Rik	4.593837426	0.006149825
1450791_at	Nppb	4.610263947	0.006060208
1451524_at	Fbxw2	4.619741472	0.006009196
1426458_at		4.621344819	0.006000616
1431716_at	Herc4	4.621895613	0.005997671
1421257_at	Pigb	4.628246541	0.005963844
1425285_a_at	Rab27a	4.63066177	0.005951038
1425709_at	Rnf17	4.684098899	0.005675746
1418460_at	Sh3d19	4.750024695	0.005356347
1434674_at	Lyst	4.776236981	0.005235221
1422567_at	Niban	4.818450492	0.005046817
1423154_at	BC005537	4.830514081	0.00499444
1425837_a_at	Ccrn4l	4.886163806	0.004760904
1419929_at		4.934007639	0.004570291
1427285_s_at	2210401K01Rik	4.971430915	0.004427403
1424841_s_at	Rbks	4.999229746	0.004324644
1456511_x_at	Eras	5.002953677	0.004311091
1460464_at	2700089E24Rik	5.011990225	0.004278412
1435106_at	3732412D22Rik	5.067838157	0.004082764
1420605_at	Mtag2	5.106047884	0.003954942
1438403_s_at	Ramp2	5.1302574	0.003876383
1438824_at	Slc20a1	5.153244671	0.003803479
1422986_at	Esrrb	5.181240688	0.003716852
1437867_at		5.202674306	0.003652093
1451416_a_at	Tgm1	5.218518597	0.003605072
1455930_at		5.274806147	0.003443681
1422903_at	Ly86	5.294879889	0.003388188
1420947_at	Atrx	5.29861609	0.003377976
1426267_at	Zbtb8os	5.321555804	0.003316062
1420946_at	Atrx	5.339806851	0.003267753
1443949_at	Ppp2r5e	5.375119299	0.003176613
1418189_s_at		5.379928117	0.003164434
1427408_a_at	Thrap3	5.479919443	0.002923217
1418188_a_at		5.510099766	0.002854707
1416325_at	Crisp1	5.528967926	0.002812835
1423411_at	BC013481	5.560493925	0.002744476
1449167_at	Epb4.1l4a	5.632515885	0.002595515
1417755_at	Topors	5.656705105	0.002547636
1424786_s_at	Wdr45	5.706240554	0.002452797
1417548_at	Sart3	5.718574747	0.002429833
1420781_at	Etos1	5.757749264	0.002358561
1425019_at	Ubxd4	5.830010327	0.002233456
1420909_at	Vegfa	5.932031625	0.002069901
1450051_at	Atrx	5.978799312	0.001999675
1420169_at		6.10858383	0.001819049
1447984_at	Gpatc2	6.132886435	0.001787407
1422259_a_at	Ccr5	6.259509495	0.001632696
1442566_at	Jarid2	6.267516862	0.001623457
1437534_at		6.415989061	0.001462848
1428786_at	4930568P13Rik	6.897342617	0.001057372
1418350_at	Hbegf	8.389351415	0.00043149
1449898_at	1-Sep	9.133148915	0.00029058

Top 200 differentially expressed genes in incorporating stem cells relative to non-incorporating stem cells.

Table 4 Top gene ontology (proliferation)

Categories	Diseases or functions annotation	P value	No. of molecules
Cellular growth and proliferation	Proliferation of cells	1.08E-08	114
Cellular development, cellular growth and proliferation	Proliferation of stem cells	5.45E-05	11
Cellular development, cellular growth and proliferation, embryonic development, development	Proliferation of embryonic cells	4.41E-04	13

The category and sub-categories annotated as “proliferation”. Shown are the top gene ontology results (P < 10E-4, number of molecules ≥ 5) of analysis performed on the 391 genes found to be significantly different between groups (P < 0.02). The analysis was performed with IPA (http://www.ingenuity.com/products/ipa, October 2014).