Unknown,Transcriptomics,Genomics,Proteomics

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LncRNA Sequencing Analysis of Mouse spermatogonial stem cells during Glial cell-derived neurotrophic factor?GDNF?treatment

ABSTRACT: Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides and have little or no potential for translation. More and more studies have domenstrated mammalian lncRNAs are intrinsically functional and growing data indicate lncRNAs are determinants of stem cell fate by regulating potency, self-renewal and differentiation. There is no report of lncRNAs in spermatogonial stem cells?SSCs?, and importance of lncRNAs in germline linage stem cell has not been investigated yet. here, we presents a large –scale profiling of all lncRNAs in SSCs as well as those lncRNAs regulated by SSC dependent growth factor GDNF through high throughput sequencing. Spermatogonial stem cells were first suffered from an 18 hr GDNF withdrawal followed by refreshment with GDNF for 8 hrs and RNA samples were collected from normal culture wells (N), GDNF 18hr withdrawal wells (0hr), and GDNF 8 hr refreshed cells (8hr). After all GDNF treatment, cultured germ cell clumps were gently blowed with a 200?l pipette, and followed by Total RNA isolation and sequencing by Illumina HiSeqTM 2000

ORGANISM(S): Mus musculus

SUBMITTER: wang Min

PROVIDER: E-GEOD-66998 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Insight into mechanisms controlling gene expression in the spermatogonial stem cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In this study we explored the global gene expression profiles of glial cell line-derived neurotrophic factor (GDNF) regulated transcription factors, Ets variant gene 5 (Etv5), B-cell CLL/lymphoma 6, member B (Bcl6b) and POU domain, class-3 transcription factor-1 (Pou3f1). We reasoned that these three factors may function as a core-set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient short-interfering RNA oligonucleotides (siRNA) to individually target Etv5, Bcl6b and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. While there were only modest overlaps in the target genes regulated by the three factors, ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes including, Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury and CXC chemokine Receptor, type 4 (Cxcr4), and we show that ETV5 binding to the Brachyury gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury significantly reduced the number of donor cell-derived colonies formed within recipient mouse testes. These results suggest Brachury is of biological importance, and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro. Microarray gene expression analysis was conducted with Affymetrix Mouse 430 2.0 GeneChips (Affymetrix Inc.).Following with gene knockdown, total RNA from spermatogonial stem cells was converted to cDNA. There are total 16 samples (Four groups and four samples per group) Negative control (N), Bcl6b Knockdown (B), Etv5 knockdown (E), and Pou3f1 knockdown (O), respectively.

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LncRNA Sequencing Analysis of Mouse spermatogonial stem cells during Glial cell-derived neurotrophic factor?GDNF?treatment

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