Overexpression Nanog Activates Pluripotent Genes in Porcine Fetal Fibroblasts and Nuclear Transfer Embryos.код для вставкиСкачать
THE ANATOMICAL RECORD 294:1809–1817 (2011) Overexpression Nanog Activates Pluripotent Genes in Porcine Fetal Fibroblasts and Nuclear Transfer Embryos LI ZHANG, YI-BO LUO, GERELCHIMEG BOU, QING-RAN KONG, YAN-JUN HUAN, JIANG ZHU, JIAN-YU WANG, HUI LI, FENG WANG, YONG-QIAN SHI, YAN-CHANG WEI, AND ZHONG-HUA LIU* Department of Life Science, Northeast Agriculture University, Heilongjiang Province, China ABSTRACT Nanog as an important transcription factor plays a pivotal role in maintaining pluripotency and in reprogramming the epigenome of somatic cells. Its ability to function on committed somatic cells and embryos has been well deﬁned in mouse and human, but rarely in pig. To better understand Nanog’s function on reprogramming in porcine fetal ﬁbroblast (PFF) and nuclear transfer (NT) embryo, we cloned porcine Nanog CDS and constructed pcDNA3.1 (þ)/Nanog and pEGFP-C1/Nanog overexpression vectors and transfected them into PFFs. We studied the cell biological changes and the expression of Nanog, Oct4, Sox2, Klf4, C-myc, and Sall4 in transfected PFFs. We also detected the development potential of the cloned embryos harboring Nanog stably overexpressed ﬁbroblasts and the expression of Oct4, Sox2, and both endogenous and exogenous Nanog in these embryos. The results showed that transient overexpression Nanog in PFF could activate the expression of Oct4 (5-fold), C-myc (2fold), and Sall4 (5-fold) in somatic cells, but they could not be maintained during G418 selection. In NT embryos, although Nanog overexpression did not have a signiﬁcant effect on blastocyst development rate and blastocyst cell number, it could signiﬁcantly activate the expression of endogenous Nanog, Oct4, Sox2 to 160-fold, 93-fold, and 182-fold, respectively (P < 0.05). Our results demonstrate that Nanog could interact with and activate other pluripotent genes both in PFFs and embryos. Anat Rec, C 2011 Wiley-Liss, Inc. 294:1809–1817, 2011. V Key words: pig; Nanog; overexpression; pluripotent genes; activation INTRODUCTION Nanog is a novel pluripotent gene that plays a crucial role in maintaining the undifferentiated state of mouse embryonic stem cells (mESCs) and inner cell mass (Chambers et al., 2003; Mitsui et al., 2003). We all know there are three ways to erase the developmental programming of differentiated cell nuclei, nuclear transfer (NT) into enucleated oocytes, cell fusion with pluripotent stem cells or introduction of combination deﬁned transcriptional factors into somatic cells. In generating induced pluripotent stem cells (iPSCs), Nanog is not included in the classic four factors (Oct4, Sox2, Klf4, and C-myc), but it plays important role in reprogramming by interacting with these pluripotent-associC 2011 WILEY-LISS, INC. V Additional Supporting Information may be found in the online version of this article. Li Zhang and Yi-Bo Luo contributed equally to this work. Grant sponsor: The National Basic Research Program of China; Grant number: 2009CB94100; Grant sponsor: National Natural Science Foundation of China; Grant number: 30871431. *Correspondence to: Zhong-Hua Liu, Department of Life Science, Northeast Agriculture University, Heilongjiang Province, China. Fax: þ86045 155191747. E-mail: firstname.lastname@example.org Received 18 April 2011; Accepted 12 June 2011 DOI 10.1002/ar.21457 Published online 3 October 2011 in Wiley Online Library (wileyonlinelibrary.com). 1810 ZHANG ET AL. ated genes. Actually, Nanog with Lin28 could replace Klf4 and C-myc then directly participate in reprogramming human somatic cells (Yu et al., 2007; Choi et al., 2009; Hanna et al., 2009). The activation of endogenous Nanog is indispensable for generating iPSCs that had the ability to contribute to adult chimeras (Takahashi and Yamanaka, 2006; Okita et al., 2007). Besides, Nanog is essential for transforming the dedifferentiated intermediates to ground state pluripotency in reprogramming process (Silva et al., 2008). Overexpression Nanog in ESCs could enhance the transfer of pluripotency in fusion experiment and overexpression Nanog is adequate to convert epiblast stem cells to ground state pluripotency (Silva et al., 2009). Pig is considered as a useful and meaningful animal model in therapeutic and biomedical research, such as bioreactor and xenotransplantation. ESCs have been considered as a useful tool for generating transgenic pig. However, the research of these potential applications progressed slowly, because no authentic porcine ESCs is available to date (Li et al., 2004; Brevini et al., 2007; Kim et al., 2007; Hall, 2008; Kim et al., 2010). Although three groups announced recently they had established iPSCs in pig by introduced human or mouse derived transcriptional factors (Oct4, Sox2, Klf4, and C-myc), none of them successfully applied the iPSCs in generating chimeras, which is the basic standard to deﬁne pluripotency (Esteban et al., 2009; Ezashi et al., 2009; Wu et al., 2009). Only one report showed that porcine mesenchymal stem cells (MSCs) transduced with six human transcription factors (Nanog,Oct4, Sox2, Klf4, C-myc, and Lin28) could generate chimeras and suggested the insufﬁcient endogenous Nanog expression may be responsible for the failure of generating chimeras (West et al., 2010). A recent report also mentioned that pig iPSCs generated by six genes had higher percentage of undifferentiated colonies during the early passages (Wu et al., 2009). All the work reminded us to pay attention to the Nanog’s function on reprogramming in pig, especially on the interaction with other pluripotent genes. Most of studies were merely conﬁned to Nanog expression patterns by immunoﬂuorescence and real time-PCR in porcine embryos and somatic stem cells, but the function of its role in cellular reprogramming and early embryo development by cooperating with other genes is still not deﬁned. (Blomberg et al., 2008; Goel et al., 2008; Magnani and Cabot, 2008; Hall et al., 2009). In this study, we cloned Nanog CDS and subcloned it to pcDNA3.1 (þ) and pEGFP-C1 vector. After transfecting the vectors into porcine fetal ﬁbroblast (PFF), we systematically detected the biological characteristics of these cells and the expression of Oct4, Sox2, Nanog, Klf4, C-myc, and Sall4 both in transient and stable transfected PFF. We also constructed NT embryos derived from Nanog overexpressed PFF and analyzed the development potential in vitro. The expression of the pluripotency master genes, Oct4, Sox2, and Nanog in different stages of early embryos was also detected by quantitative Real time-PCR. This research could help us to better understand the role of Nanog in cellular reprogramming and early embryos development in pig. MATERIALS AND METHODS Gene Cloning and Vector Construction The Nanog gene was ampliﬁed by reverse transcriptase-polymerase chain reaction (RT-PCR) using total RNA extracted from pools of MII stage oocytes using Tri- zol (Invitrogen). And the total RNA was reverse transcript using High Capacity cDNA Reverse Transcription Kit (ABI). The PCR ampliﬁcation was carried out for one cycle with denaturing at 94 C for 4 min, and 35 subsequent cycles with denaturing at 95 C for 30 s, annealing at 56.5 C for 30 s, extension at 72 C for 30 s, and a ﬁnal extension at 72 C for 10 min. The sense primers 50 -AT GAGTGTGGATCCAGCTTGTC-30 and antisense primer 50 -TCACATATCTTCAGGCTGTATG-30 are used for Nanog cloning. To construct the pcDNA3.1 (þ)/Nanog plasmid, the Nanog cDNA was insert into pcDNA3.1 (þ) (Invitrogen) between HindIII and XhoI. Nanog cDNA was also inserted into pEGFP-C1 (Invitrogen) between BglII and XhoI to generate pEGFP-C1/Nanog that expresses GFP-Nanog fusion protein. Cell Culture and Transfection Pig Fetal Fibroblasts (PFF) were isolated from 35-daysold male fetus, which were dissected by scalpel blade in PBS buffer and then treated in collagenase/DNAse I at 37 C for 45 min. The solution was mixed with PFF culture medium: DMEM (high glucose) supplemented with 10% FBS and penicillin/streptomycin (all the reagents are from Gibco), centrifuged at 1,000 rpm for 5 min and suspended in PFF culture medium. The cells were passaged in PFF culture medium without any antibiotics. For transfection experiment, cells were plated in a 3.5-cm plate to achieve 80–90% conﬂuence within 24 h in PFF culture medium without penicillin/streptomycin. Cells were transfected with Lipofactamine 2000 (Invitrogen) according the instruction with a ratio of 3:1 transfection reagent (ml): DNA (mg). Cells were passaged 24 h after transfection. Selection medium DMEM (high glucose supplemented with 10% FBS and 500 ug/mL G418) replaced the PFF culture medium after cells adhesion. Cells for stable selection were rendered by G418 selection for 20 days, when the untransfected cells died completely, while cells for transient transfection were harvested at 24 h later after transfection. Immunoﬂuorescence Porcine ﬁbroblasts were seeded and cultured for up to 3 days before analysis. Growth medium was removed, and the cells were ﬁxed with 4% Para formaldehyde/PBS (pH 7.4) for at least 40 min, followed by permeabilization with 1% TritonX-100 at room temperature for 30 min, blocked in 1% BSA supplemented PBS for 1 h and then incubated with rabbit anti-Nanog antibody (Santa Cruz; 1:200) overnight at 4 C. After three washes with PBS containing 0.1% Tween 20 and 0.01% Triton X-100 for 5 min each, ﬁbroblast cells were labeled with 1:200 FITC-conjugated IgG for 1 h at room temperature. After washing in PBS containing 0.1% Tween 20 and 0.01% Triton X-100, the cells were co-stained with Hoechst33342 (10 mg/mL in PBS) and imaged on a Nikon Eclipse 80i microscope. Oocytes In-Vitro Maturation Pig (Sus scrofa) ovaries from prepubertal gilts were collected at a local slaughterhouse and transported to the laboratory in an insulated container at 37 C. Antral follicles between 5 and 8 mm in diameter were aspirated manually with a disposable 10-cc syringe and an 18-gauge NANOG ACTIVATES PORCINE GENES EXPRESSION needle. Follicular ﬂuid was pooled and allowed to settle by gravity. Cumulus-oocytes complexes (COCs) were resuspended in Hepes-buffered medium containing 0.01% polyvinyl alcohol (PVA). Under a dissecting microscope, COCs with multiple layers of intact cumulus cells were selected for the experiments. Around 50–75 COCs were placed in 500 mL of tissue culture medium 199 (TCM-199; Gibco BRL, Grand Island, NY) containing 0.14% PVA, 10-ng/mL epidermal growth factor, 0.57-mM cysteine, 0.5-IU/mL pig FSH, and 0.5-IU/mL ovine LH. COCs were matured for 42–44 h at 39 C and 5% CO2 in air, 100% humidity. All chemicals were obtained from Sigma Chemical Company (St. Louis, MO) unless stated otherwise. In Vitro Fertilization, NT and Embryos Culture Following short time incubation at 39.5 C, the extended semen was resuspended with DPBS (0.1% BSA), and washed 2 times with DPBS (0.1% BSA) by centrifugation at 2,000 rpm for 4 min. The spermatozoa concentration was measured using a hemocytometer and the proportion of motile sperm was determined. The spermatozoa were diluted with mTBM to optimal concentration. Cumulus-free matured oocytes were washed three times in mTBM IVF medium. Approximately, 30– 35 oocytes were transferred into 50-mL droplets of IVF medium covering with mineral oil that had been equilibrated for 30 min at 39.5 C in 5% CO2 in air before adding the sperm. 50-mL sperm sample was added to the fertilization droplets containing oocytes, giving a ﬁnal sperm concentration of 6 104 cells/mL. Oocytes were coincubated with sperm in mTBM for 5 h. Then the oocytes were washed and cultured in PZM-3. The in-vitro mature oocytes were enucleated by aspirating the ﬁrst polar body and the adjacent cytoplasm in manipulation droplet (TCM-199-HEPES enriched with 0.3% BSA). Nanog stably overexpressed PFF were used as donor cells and were placed under the zona pellucida of enucleated oocytes. Fusion/activation was induced by successive DC impulse of 1.2 kv/cm for 30 ls. Constructed embryos were cultured in PZM-3 at 39 C under 5% CO2 in air. The blastocysts were collected at 156 h, and then were stained in DPBS enriched with 10 mg/L Hoechst33342 in dark. After staining, the blastocysts were transferred to slides with 7-uL glycerol on it, covering with cover glass and mounting by nail polish. The wavelength of 488 nm was used to detect whether the blastocysts were GFP positive. 1811 Quantitative Real-Time Polymerase Chain Reaction Real-time PCR was performed using the SYBR Premix Ex TaqTM (TaKaRa) and the 7300 Real-Time PCR System (Applied Biosystems), with the following parameters: 95 C for 10 s, followed by 40 cycles at 95 C for 5 s and at 61 C for 34 s. Negative controls in the PCR assay including an RT reaction are completed by the same procedure with no templates. For each cDNA sample, both target and reference genes were ampliﬁed independently on the same plate and in the same experimental run in triplicate. Statistical Analysis The levels of gene expression among three groups were analyzed using analysis of variance (ANOVA) and t-test by Stat View (SAS Institute, Cary, NC); P values <0.05 were considered signiﬁcantly different. RESULTS Cloning of Porcine Nanog CDS and Construction of pcDNA3.1 (1)/Nanog and pEGFP-C1/Nanog Vector The complete cDNA of Nanog was cloned from RNA of MII stage oocytes by RT-PCR. Conﬁrmed by DNA sequencing, CDS of Nanog was composed of 915 nucleotides, and shared 99.8% identity with porcine Nanog in NCBI (GenBank accession no.FJ882402.1), and there are two nucleotide substitutions at 562 (G to A) and 572 (A to G) site. The alignment showed that the similarities of Nanog CDS sequence of pig with human, mouse, rat, bovine, goat and monkey were 80.3, 72.4, 72.2, 86.0, 86.7, and 79.5%, and the similarities of amino acid sequence were 72.9, 60.5, 61.0, 81.9, 83.3, and 72.6%, respectively. The homeodomain shared 100% identity with porcine Nanog in NCBI, 98% identity with bovine and goat, and the lowest identity with mouse and rat. The ampliﬁed PCR product was subcloned into the pcDNA3.1 (þ) and pEGFP-C1 to produce the pcDNA3.1 (þ)/Nanog (PC-Nanog), pEGFP-C1/Nanog (PG-Nanog) vectors. The Blast results of CDS and encoded amino acids from different species were present in Supporting Information Figure 1. Successful Expression of Exogenous Nanog in PFF RNA Isolation and Reverse Transcription Total RNA was extracted from pools of MII oocytes (100–200) using the Absolutely RNA Microprep Kit (Stratagene). Total RNA was extracted from pools of embryos (40–80) at the following stages: 2-cell, 4-cell, 8cell, morula and blastocyst. Oocytes and embryos were washed three times in PVA-PBS (nuclease free) and lysed in 100-lL lysis buffer (Stratagene), and then stored at 80 C until total RNA isolation. High-Capacity cDNA Reverse Transcription Kit (ABI) was used for gene cloning. PrimeScriptV RT reagent Kit was used to perform Reverse transcription for Real time-PCR according to manufacturer’s instructions (TaKaRa). R We used PG-Nanog to express GFP-NANOG fusion protein to conﬁrm the expression and the localization of exogenous Nanog. After transient transfection, PFF were transformed from the normal phenotype into proliferative phenotype that the shape of cells became round and small (Fig. 1B). We found the GFP-Nanog was localized in the nuclei of both stably and transiently transfected PFF (Fig. 1C,D). Real time-PCR results demonstrated that Nanog mRNA was successfully expressed in transient and stable transfected PFF (Fig. 2A). Immunoﬂuorescence analysis also showed that Nanog protein was expressed in PFF stably transfected by PC-Nanog (Fig. 2B). 1812 ZHANG ET AL. Fig. 1. The morphology of transfected PFF and expression of GFPNanog fusion protein. The morphology of pcDNA3.1 (þ) transiently transfected PFF (A) and pcDNA3.1 (þ)/Nanog (PC-Nanog) transiently transfected PFF (B). GFP-Nanog fusion protein was localized in the nuclei of pEGFP-C1/Nanog (PG-Nanog) stably transfected PFF (C) and transiently transfected PFF (D). GFP expression was visualized under blue light. Both of cells in (A) and (B) were observed at 24 h after transfection without G418 selection. Activation of Pluripotent Genes by Nanog Overexpression in PFF 0.05), compared with the control group (Fig. 3A). We then analyzed the expression level of these pluripotent genes in transfected PFF after G418 selection for 20 days (Fig. 3B).Compared with the control group, Nanog expression increased by 20-fold (Fig. 2B). But the expression of Oct4, Sox2, C-myc declined signiﬁcantly (P < 0.05). The expression of Klf4 and Sall4 changed insigniﬁcantly. To clarify if Nanog can interact with other pluripotent genes in PFF, we detected the expression level of Nanog at 1, 5, 10, and 15 days post-transfection without G418 selection by Real-time PCR. Compared with the Nanog expression level in PFF, Nanog expression after transient transfection were 2664, 123, 64, 9-fold at 1, 5, 10, and 15 days, respectively. Nanog peaked at Day 1 and decreased gradually (Supporting Information Figure 2). Based on these data in our preliminary experiment, we examined the expression level of pluripotent genes Oct4, Sox2, C-myc, Klf4, and Sall4 at Day 1 after transfection when Nanog expression was at the peak. The expression level of Oct4, Sox2, Klf4, C-myc, and Sall4 were 4.7- and 5.4-fold, 1.4- and 0.9-fold, 1.4- and 1.3-fold, 2.2 and 1.6fold, 4.9- and 1.9-fold in PFF transfected by PC-Nanog and PG-Nanog, respectively. The expression level of Oct4, Sall4, and C-myc increased signiﬁcantly (P < Activation of Endogenous Oct4, Sox2, and Nanog in NT Embryos Derived from Nanog Overexpressed PFF We used pcDNA3.1 (þ) empty vector and pcDNA3.1 (þ)/Nanog vector stably transfected cells as donor cells for SCNT. All reconstructed embryos were in vitro cultured at 6.5 days, and there were no statistic differences in the fusion rate, cleavage rate, and the blastocyst rate and the blastocyst cell numbers among the three groups. NANOG ACTIVATES PORCINE GENES EXPRESSION 1813 Fig. 2. Nanog protein successfully expressed in PFF. Relative expression of Nanog in transiently transfected PFF and stably transfected PFF by PC-Nanog and PG-Nanog (A). Immunoﬂuorescence results also showed that Nanog protein was expressed in PFF stably transfected by PC-Nanog. Nuclei were stained by Hoechst33342 (B). We analyzed Oct4, Sox2, both endogenous Nanog and whole amount of Nanog expression level by Real timePCR in NT embryos derived from Nanog overexpressed PFF (NT-Nanog). We used different primers for detecting exogenous Nanog and whole amount expression of Nanog in embryos. Exogenous Nanog expression could be obtained by the different values. We found exogenous Nanog peaked at 4-cell stage, while endogenous Nanog, Oct4 and Sox2 got to the highest level at morula stage, the transcripts increased by 159-fold, 93-fold, and 180fold, respectively (Fig. 4B). In blastocysts, all the three genes expression decreased to a very low level (Fig. 4B). As controls we also detected genes expression both in in vitro fertilized (IVF) and NT embryos. There were no obvious variation in the Oct4 and Sox2 expression among the different cleavage embryos, only Nanog had a signiﬁcant increase in 8-cell embryos both in IVF and NT embryos (P < 0.05). In NT embryos, Nanog expression was signiﬁcantly lower than that in IVF embryos at morula stage (P < 0.05). DISCUSSION Transient Elevation of Nanog Expression can Activate Pluripotent Genes Expression Nanog is one of the core transcription factors involved in cellular pluripotency. Although high throughput studies showed there was a list of Nanog target genes in different types of cell line, few results provided directly data showing Nanog interacting with and regulating other genes by classical experimental approaches, especially in porcine committed cells and embryos. Oct4, Sox2, Klf4, and C-myc are the key factors both in mESCs and iPSCs. Sall4 is also an important component of the transcription regulatory net works in ES cells by cooperating with Nanog and plays positive roles in the generation of pluripotent stem cells from blastocysts and ﬁbroblasts (Wu et al., 2006; Tsubooka et al., 2009). All the endogenous genes we looked at in this study have direct or indirect relations with Nanog. Results in this study clearly showed ectopic expressed porcine Nanog in PFF could upregulate Oct4, C-myc, and Sall4 but could not active Sox2 and Klf4. It indicated that porcine Nanog had similar properties in PFF, at least partially, which were described for Nanog in primate and mouse ESCs. In both humans and mice, research shows that Nanog could bind to the upstream element of Oct4 and cooperate with other core transcription factors to activate Oct4 expression thereby maintaining the pluripotency (Boyer et al., 2005; Rodda et al., 2005; Pan and Thomson, 2007). In mESCs, Nanog interacts with Sall4 in a way similar with that of Oct4 and Sox2 to activate each other, thus helps to set up the pluripotency regulatory circuits (Wu et al., 2006). It is also found that C-Myc is a downstream factor of Nanog (Loh et al., 2006). Klf4 could promote cell proliferation by suppressing p53 expression. Since p53 is a negative regulator of Nanog, Klf4 may be an upstream factor in the activation of Nanog during reprogramming by suppressing p53 expression (Zhang et al., 2010). So 1814 ZHANG ET AL. transient transfection of Nanog in our study could not activate Klf4 directly. However, it is unexpected that Nanog could activate Sox2 in NT embryos but could not upregulate Sox2 expression in PFF. One possibility is that Nanog shares different targets between porcine embryos and ﬁbroblasts. Microarray analysis conﬁrms that there is very little overlap of Nanog’s target genes in cell lines derived from different tissues, suggesting that Nanog’s target genes are variable in a tissue speciﬁc manner (Piestun et al., 2006). The genome is demethylated in ESCs and embryos, so Nanog is prone to occupy its target sites in many promoters (Boyer et al., 2005). Another possible explanation is that Nanog is not enough to activate Sox2 in PFF, but along with additional transcription factors existed in embryos to activate Sox2 successfully. Pluripotent Genes Expression Cannot be Maintained During G418 Selection Fig. 3. Relative expression of pluripotent-related genes after transient and stable transfection by PC-Nanog and PG-Nanog in PFF. Genes expression in transiently transfected PFF was detected at 24 h after transfection and in stably transfected PFF with G418 selection for 20 days. Fold difference was calculated with respect to the PFF transfected by pcDNA3.1 (þ) empty vector. Bars represent the means standard errors of three independent experiments. In our experiment, after 20 days’ G418 selection, the expression of Oct4, C-myc and Sall4 gradually silenced, although Nanog could still be maintained at comparatively high level. We speculate that transient high Nanog expression can activate part of the pluripotency related genes in PFF, but it is not enough for inducing PFF to a fully pluripotent state, thus cannot set up the stable regulatory circuit. Pan et al. also indicate that the key pluripotent factors always work together to control the whole set of pluripotency machinery rather than work individually (Pan and Thomson, 2007). So in a partially transformed PFF, these activated pluripotent genes can be easily silenced in many ways including DNA highly methylating. And C-myc as the target transcriptional factors of Nanog has the function in loosing chromosome structure, which makes many transcriptional factors prone to Fig. 4. Constructed embryos using PFF, PC and PC-Nanog stably transfected PFF as donor cells develop to blastocysts in vitro (40) (A). Relative expression of endogenous Nanog, whole amount of Nanog, Oct4 and Sox2 in porcine IVF, NT and NT-Nanog embryos at different stages (B). Nanog histogram height of green bars represents total Nanog mRNA relative expression; Histogram height of purple bars represents endogenous Nanog expression. Exogenous Nanog expression could be obtained by the different values, which in ﬁgure 4B are the green parts. Different subscripts represent signiﬁcant differences among the three kinds of embryos at the same development stages after t-test by Stat View (P values <0.05). Fold difference was calculated with respect to the MII stage oocytes. 1815 NANOG ACTIVATES PORCINE GENES EXPRESSION TABLE 1. Primers for quantitative Real time-PCR Gene Primer sequences (50 to 30 ) Product size (bp) GenBank accession no. Klf4 F:TGGGTGCGGAGGAACTGCTA R:GGGACTGACCTTGGTAATGGAGC F:CCCCTACCCGCTCAACGACA R:AGCCAAGGGTTCGGGACTGC F:GAAGGTGTTCAGCCAAACGAC R:CGATACTTGTCCGCTTTC F:AACCAGAAGAACAGCCCAGAC R:TCCGACAAAAGTTTCCACTCG F:ATCCACCTCCGCTCCCATACC R:CGTTGCCTGCCGTCATCTTGT F:AGCCTCCAGCAGATGCAAGAACTCT R:TTCTGCCACCTCTTACATTTCATTCG F:AGCCCCAGCTCCAGTTTCAGC R:AATGATCGTCACATATCTTCAGGCTGTA F:GCCCGAAGCGTTTACTTTGA R:CCGCGGTCCTATTCCATTATT 153 NM_001005154.1 133 NM_001005154.1 185 NM_001113060.1 155 NM_001123197.1 166 NM_001114673.1 181 FJ882402.1 104 FJ882402.1 C-myc Oct4 Sox2 Sal14 Nanog EndoNanog 18SrRNA 93 NR_002170.3 TABLE 2. Development potential of NT embryos produced by different donor cells Donor cells pCDNA transfected PEF pCDNA3.1(þ)/Nanog transfected PEF PEF(F-7) No. of embryos produced No. of embryos fused (% SD) No. of embryos cleaved (% SD) No. of blastocysts (% SD) Nuclear numbers of blastocysts 242 243 224 168(70.2 14.9) 210(86.4 4.3) 168(75.1 9.6) 67(40.2 11.6) 83(39.1 20.2) 86(43.8 22.8) 21(12.1 5.0) 28(14.1 3.4) 33(17.8 7.9) 49 4.6 50 5 67 2.6 binding to the genome DNA site. Following with Nanog expression decreased intensively, C-myc has an extraordinary reduction in PFF, which made other pluripotent genes (Oct4, Sox2, and Nanog) difﬁcult to bind to the target site in condensed chromosomes. As a result, the transcription factors are degraded and represent signiﬁcant reduction. That might be the reason that Oct4, Sox2, and C-myc were inhibited in Nanog stable expressed PFF. Exogenous Nanog Activates Endogenous Oct4, Sox2, and Nanog Expression in NT Embryos Nanog is a critical regulator for embryonic development and plays an important role in lineage differentiation by cooperating with Oct4, which has been deﬁned in mouse, but are still not clearly understood in pig (Strumpf et al., 2005; Messerschmidt and Kemler, 2010). Immunoﬂuorescence studies demonstrate that Nanog expression cannot be detected in in vitro porcine blastocysts and Oct4 is not conﬁned to the ICM, which is not identical with the studies in mouse (Kuijk et al., 2008). In this study, we ﬁnd that there is expression of Nanog, Oct4, and Sox2 both in IVF and NT porcine embryos from 2-cell to morula stage, which keeps a comparatively stable level except there is an elevation of Nanog at 8cell stage. However, all of these three genes expression is downregulated at blastocyst stage, which is partially coincident with the previous reports (Magnani and Cabot, 2008; Xing et al., 2009). Although overexpression Nanog in transgenic NT embryos could activate endogenous Nanog, Oct4, and Sox2 expression and the level is much higher than that is in IVF and normal NT embryos, these genes expression is still downregulated at blastocyst stage. Combining with the result that the expression of all these three genes have no inﬂuence on the embryos’ development in vitro which is assessed by blastocyst rate and blastocyst cell number, we speculate that Nanog cooperating with Sox2 and Oct4 may function as a key regulating factor in ICM/trophoblast and epiblast /hypoblast differentiation at a relatively late stage of porcine blastocysts compared with mouse embryos. After all, there exists a long spanning blastocyst stage and different developmental process in porcine embryos. And a recent study also demonstrated that Nanog was detected later in the epiblast and hypoblast of spherical blastocysts of pig (Wolf et al., 2011). But in early developmental stages before morula, it must play other regulatory roles different with that in the later stage, which is still needed to be identiﬁed. Formation of ES-like Colony Observed in Nanog Overexpressed PFF Another interesting phenomenon in our study is that we observed the formation of ES-like colonies (Supporting Information Figure 3). Both sizes and shapes of these colonies were not disciplinary with unclear cell boundaries. By removal of these colonies, the rest of the basal cells could regenerate colonies through passaging. But after 15–20 days, it would not reoccur. To exclude the possibility that transfection and followed prolonged antibiotic selection cause considerable stress on the cultured cells, we also ran the control experiments that transfection with pcDNA3.1(þ) empty vector in PFF, but we did not ﬁnd the colonies that we observed in PFF transfected either by pcDNA3.1(þ)/Nanog or by pEGFPC1/Nanog. Previous studies point that overexpression Nanog in ﬁbroblasts results in increased cell proliferation and transformation of foci-forming phenotype (Zhang et al., 2005). Although overexpression Nanog in 1816 ZHANG ET AL. human ESCs and MSCs can enhance colony formation (Darr et al., 2006; Liu et al., 2009). These results indicate that in PFF the porcine Nanog can promote colony formation as in human ESCs and MSCs, other than just promote cell proliferation that also happens in other species ﬁbroblast. Actually, we examined changes in cell doubling time in Nanog transfected cells and the results showed that Nanog could also promote the proliferation of PFF (Supporting Information Figure 4). We presume this phenomenon caused by Nanog overexpression may be also associated with LIF/Stat3 signal pathway, because our results showed there was expression elevation of LIF, LIFr, Gp130, and Stat3 in the Nanog transient overexpression PFF (Supporting Information Figure 5). Primers are shown in Supporting Information Table 1. All the information strongly suggests that Nanog should be one of the key factors for porcine iPSCs induction. ACKNOWLEDGEMENTS The authors are grateful to Jun-Yu Ma at Northeast Agricultural University for his valuable discussions. The authors are also thankful to colleagues in the ‘‘Lab of Embryo Biotechnology’’ for their helpful discussions. LITERATURE CITED Blomberg LA, Schreier LL, Talbot NC. 2008. Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast during in vitro culture. Mol Reprod Dev 75:450–463. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. 2005. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122:947– 956. Brevini TA, Antonini S, Cillo F, Crestan M, Gandolﬁ F. 2007. Porcine embryonic stem cells: facts, challenges and hopes. Theriogenology 68(Suppl 1):S206–S213. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643–655. Choi KD, Yu J, Smuga-Otto K, Salvagiotto G, Rehrauer W, Vodyanik M, Thomson J, Slukvin I. 2009. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells 27:559–567. Darr H, Mayshar Y, Benvenisty N. 2006. Overexpression of NANOG in human ES cells enables feeder-free growth while inducing primitive ectoderm features. Development 133:1193–1201. Esteban MA, Xu J, Yang J, Peng M, Qin D, Li W, Jiang Z, Chen J, Deng K, Zhong M, Cai J, Lai L, Pei D. 2009. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J Biol Chem 284:17634–17640. Ezashi T, Telugu BP, Alexenko AP, Sachdev S, Sinha S, Roberts RM. 2009. Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci USA 106:10993–10998. Goel S, Fujihara M, Minami N, Yamada M, Imai H. 2008. Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis. Reproduction 135:785–795. Hall V. 2008. Porcine embryonic stem cells: a possible source for cell replacement therapy. Stem Cell Rev 4:275–282. Hall VJ, Christensen J, Gao Y, Schmidt MH, Hyttel P. 2009. Porcine pluripotency cell signaling develops from the inner cell mass to the epiblast during early development. Dev Dyn 238:2014–2024. Hanna J, Saha K, Pando B, van Zon J, Lengner CJ, Creyghton MP, van Oudenaarden A, Jaenisch R. 2009. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462:595– 601. Kim HS, Son HY, Kim S, Lee GS, Park CH, Kang SK, Lee BC, Hwang WS, Lee CK. 2007. Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts. Zygote 15:55–63. Kim S, Kim JH, Lee E, Jeong YW, Hossein MS, Park SM, Park SW, Lee JY, Jeong YI, Kim HS, Kim YW, Hyun SH, Hwang WS. 2010. Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts. Zygote 18:93–101. Kuijk EW, Du Puy L, Van Tol HT, Oei CH, Haagsman HP, Colenbrander B, Roelen BA. 2008. Differences in early lineage segregation between mammals. Dev Dyn 237:918–927. Li M, Li YH, Hou Y, Sun XF, Sun Q, Wang WH. 2004. Isolation and culture of pluripotent cells from in vitro produced porcine embryos. Zygote 12:43–48. Liu TM, Wu YN, Guo XM, Hui JH, Lee EH, Lim B. 2009. Effects of ectopic Nanog and Oct4 overexpression on mesenchymal stem cells. Stem Cells Dev 18:1013–1022. Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, Bourque G, George J, Leong B, Liu J, Wong KY, Sung KW, Lee CW, Zhao XD, Chiu KP, Lipovich L, Kuznetsov VA, Robson P, Stanton LW, Wei CL, Ruan Y, Lim B, Ng HH. 2006. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38:431–440. Magnani L, Cabot RA. 2008. In vitro and in vivo derived porcine embryos possess similar, but not identical, patterns of Oct4, Nanog, and Sox2 mRNA expression during cleavage development. Mol Reprod Dev 75:1726–1735. Messerschmidt DM, Kemler R. 2010. Nanog is required for primitive endoderm formation through a non-cell autonomous mechanism. Dev Biol 344:129–137. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S. 2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631–642. Okita K, Ichisaka T, Yamanaka S. 2007. Generation of germlinecompetent induced pluripotent stem cells. Nature 448:313–317. Pan G, Thomson JA. 2007. Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res 17:42–49. Piestun D, Kochupurakkal BS, Jacob-Hirsch J, Zeligson S, Koudritsky M, Domany E, Amariglio N, Rechavi G, Givol D. 2006. Nanog transforms NIH3T3 cells and targets cell-type restricted genes. Biochem Biophys Res Commun 343:279–285. Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P. 2005. Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 280:24731–24737. Silva J, Barrandon O, Nichols J, Kawaguchi J, Theunissen TW, Smith A. 2008. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol 6(10):e253. doi:10.1371/ journal.pbio.0060253. Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O, Wray J, Yamanaka S, Chambers I, Smith A. 2009. Nanog is the gateway to the pluripotent ground state. Cell 138:722–737. Strumpf D, Mao CA, Yamanaka Y, Ralston A, Chawengsaksophak K, Beck F, Rossant J. 2005. Cdx2 is required for correct cell fate speciﬁcation and differentiation of trophectoderm in the mouse blastocyst. Development 132:2093–2102. Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult ﬁbroblast cultures by deﬁned factors. Cell 126:663–676. Tsubooka N, Ichisaka T, Okita K, Takahashi K, Nakagawa M, Yamanaka S. 2009. Roles of Sall4 in the generation of pluripotent stem cells from blastocysts and ﬁbroblasts. Genes Cells 14:683– 694. West FD, Terlouw SL, Kwon DJ, Mumaw JL, Dhara SK, Hasneen K, Dobrinsky JR, Stice SL. 2010. Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells Dev 19:1211–1220. Wolf XA, Serup P, Hyttel P. 2011. Three-dimensional localisation of NANOG, OCT4, and E-CADHERIN in porcine pre- and peri-implantation embryos. Dev Dyn 240:204–210. NANOG ACTIVATES PORCINE GENES EXPRESSION Wu Q, Chen X, Zhang J, Loh YH, Low TY, Zhang W, Sze SK, Lim B, Ng HH. 2006. Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells. J Biol Chem 281:24090–24094. Wu Z, Chen J, Ren J, Bao L, Liao J, Cui C, Rao L, Li H, Gu Y, Dai H, Zhu H, Teng X, Cheng L, Xiao L. 2009. Generation of pig induced pluripotent stem cells with a drug-inducible system. J Mol Cell Biol 1:46–54. Xing X, Magnani L, Lee K, Wang C, Cabot RA, Machaty Z. 2009. Gene expression and development of early pig embryos produced by serial nuclear transfer. Mol Reprod Dev 76:555–563. 1817 Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin, II, Thomson JA. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920. Zhang J, Wang X, Chen B, Suo G, Zhao Y, Duan Z, Dai J. 2005. Expression of Nanog gene promotes NIH3T3 cell proliferation. Biochem Biophys Res Commun 338:1098–1102. Zhang P, Andrianakos R, Yang Y, Liu C, Lu W. 2010. Kruppel-like factor 4 (Klf4) prevents embryonic stem (ES) cell differentiation by regulating Nanog gene expression. J Biol Chem 285:9180– 9189.