they arise [13,14]. Genes encoding the imprinted miRNAs are mainly clustered in two chromosomal domains [PWS-AS (also called Snurf-Snrpn) cluster and Dkl1-Gtl2 cluster] in mouse although few single imprinted miRNA are also present at several genomic regions [14,15,16,17,18,19]. Furthermore, almost all well-charac- terized imprinted genes clusters such as Igf2-H19, Peg10, Copg2, Rasgfr1, Gnas-Nespas, Kcnq1 and Igf2r-Air also encode one or more imprinted miRNAs whose expression is restricted in a parent-of- origin manner and is controlled by DNA methylation at imprinting control region (ICR) of the respective gene cluster [14,15,16]. These imprinted miRNAs show distinct temporal- and tissue-specific expression patterns in different tissues, including ES cells, and control a wide range of developmental and physiological pathways, including stem cell pluripotency and differentiation [20,21,22,23,24,25,26]. Recent studies have shown that maGS and ES cells have similar miRNA profile [27,28]. However, miRNA profile of GS cell has not been investigated. We recently showed that GS and maGS cells show differential expression of Let-7 and miR-294 miRNAs which may serve as miRNA signature to distinguish GS cells from maGS or ES cells [29]. However, nothing is known in literature about genomic imprinting or expression of imprinted miRNA in GS and maGS cells. Recent studies have also shown that, a cluster of imprinted miRNA encoded by imprinted Dlk1-Dio3 locus correlates with the pluripotency levels of mouse stem cells [22] and consequently, imprinting status of this locus can serve as a marker to identify fully pluripotent iPS (induced pluripotent stem cell) or ES cells from partial pluripotent cells [22,25]. However, genomic imprinting and expression of imprinted miRNAs, including those encoded by Dlk1-Dio3 locus, in testes-derived male germ-line stem cells are not known. Therefore, we extended our earlier study [29], to investigate the genomic imprinting and expression of a selected number of imprinted miRNAs in mouse testes-derived male germ-line stem cells. Three imprinted miRNAs, miR-127, miR-483 and miR-296, which are encoded from Dlk1-Dio3, Igf2-H19 and Gnas-Nespas imprinted gene clusters, respectively under the regulation of common ICRs (Dlk1-Dio3 IG-DMR, Igf2-H19 ICR and Gnas- Nespas DMR) for all miRNAs in the respective gene clusters were analyzed (Figure 1A) [20,30,31,32]. These imprinted gene clusters were specifically chosen because DNA methylation at both Dlk1- Dio3 IG-DMR and Igf2-H19 ICR is imprinted (i.e., DNA methylated) on paternal chromosome to suppress the expression of miR-127 and miR-483 from paternal and maternal chromo- some, respectively. On the other hand, Gnas-Nespas DMR is imprinted (i.e., DNA methylated) on maternal chromosome to suppress the expression of miR-296 from maternal chromosome [14,15,30]. Thus, analysis at these three imprinted gene clusters encompass all possible combinations of paternal imprinting controlling maternally- or paternally- expressed miRNAs and vice versa. Materials and Methods All chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) unless otherwise specifically indicated. All animal experimentations of the study were approved by the Institutional Bio-safety Committee (Approval ID Nos. : 09-01-020, 09-01-022, 09-01-025, 09-01-026, and 09-01-027) that oversees the ethical and animal experimental issues. Establishment of GS and maGS cell lines The GS and maGS cells were isolated from 4- to 6- week old DBA/2 mouse (Orient Bio, South Korea) testes as described Imprinted miRNA Signature in Male GS Cells earlier with partial modifications [5,6,29]. The isolated cells were cultured on mitomycin C- treated STO feeder cell layer in Dulbecco’s modified Eagle medium (DMEM) supplemented with 15% (v/v) fetal bovine serum (Hyclone, Logan, UT), MEM nonessential amino acids (Gibco BRL, Grand Island, NY), 1% penicillin-streptomycin (Gibco BRL), 50 mM 2-mercaptoethanol, and 10 ng/ml recombinant human GDNF (R & D Systems, Minneapolis, MN) at 37uC in a humidified atmosphere of 5% CO2 in air. Additionally, 1000 units/ml murine leukemia inhibiting factor (LIF; Chemicon, Temecula, CA) was added to initial culture medium to enhance the formation of germ cell colonies [33]. The GS and maGS cell colonies were subsequently cultured in the absence (GS) or presence (maGS) of LIF to establish the respective cell lines [5,6]. The established maGS cell lines were subsequently maintained in feeder-free conditions while GS cell lines were cultured on mitomycin C- treated STO feeder cell layer. The characteristics of established GS and maGS cell lines were verified by expression of marker genes and alkaline phosphatase (AP) activity essentially as we described previously [10,11] (Supplementary Figure S1). The maGS cells, cultured under the current culture conditions, resembled the ES cells for the hemi-zygotic DNA methylation pattern of Igf2-H19 ICR and the expression of Let-7 and miR-294 miRNAs (Supplementary Figure S2). For in vitro differentiation studies, GS and maGS cells were induced to form embryoid bodies (EB) by hanging drop culture for four days and then cultured in the presence of 1.0 mM all-trans retinoic acid (EBRA) for next four days as we described earlier [10,11]. As control, mouse ES cells were cultured for analysis by standard method [34]. Analyses of miRNAs The expression of miRNAs was quantified by real-time TaqMan MicroRNA Assay essentially as we described earlier [29]. Briefly, total RNA, including miRNA, was extracted from cultured cells using the mirVanaR microRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. The isolated miRNAs (10 ng) were then converted to cDNA using TaqManR MicroRNA Reverse Transcription Kit (Ambion) in 15 ml reaction mixture containing 100 mM dNTPs, 50 U/ml MultiScribeR reverse transcriptase, 20 U/ml RNase inhibitor and 3 ml of miRNA-specific reverse transcription (RT) primer according to the manufacturer’s instructions and amplified for specific miRNA on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) using commercially available optimized miRNA-specific primers for each miRNA and for the endogenous sno202 control. The amplification parameters for RT and real-time PCR were employed according to the manufacturer’s protocol (TaqManR MicroRNA assay kit; Applied Biosystems). This method quantifies exclusively mature miRNAs but not their precursors. The expression values for each miRNAs were normalized to endogenous sno202 control and relative expression values were obtained using the 22DDCt method (Applied Biosystems user bulletin PN 4347825). All experiments were performed in triplicates. Analyses of DNA methylation DNA methylation status of GS and maGS cells was analyzed by bisulfite genomic sequencing essentially as we described earlier [10,11]. Briefly, genomic DNA (2 mg) was treated with sodium bisulfite using Epitect bisulfite kit (Qiagen, Hilden, Germany) and PCR-amplified using specific primer pairs for Dlk1-Dio3 IG-DMR, H19 ICR and Gnas-Nespas DMR (Supplementary Table S1). Amplified PCR-products were then cloned in pGEM-T Easy PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22481