Screening for Genes Essential for Mouse Embryonic Stem Cell Self‐Renewal Using a Subtractive RNA Interference Library

Zhang, Jun Zheng; Gao, Wei; Yang, Hong; Zhang, Bo; Zhu, Zuo Yan; Xue, You Fang

doi:10.1634/stemcells.2006-0017

Cited by 42 publications

(41 citation statements)

References 26 publications

(31 reference statements)

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“…5 In addition to single gene knockdown, investigators developed RNAi libraries to discover gene pathways controlling various cellular processes and facilitate global understanding of cellular behavior. 6 --9 This approach was used to identify genes that are involved in proteasome-mediated proteolysis, 10 virus infection and replication, 11 --14 cytokinesis, 8,9,15,16 endocytosis, 17 stem cell self-renewal 18 or cancer cell proliferation and survival. 19,20 RNAi knockdown requires a good selection strategy to link genetic information to cellular phenotype.…”

Section: Introductionmentioning

confidence: 99%

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

et al. 2012

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Loss of gene function is a valuable tool for screening genes in cellular processes including stem cell differentiation differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis and real-time, dynamic information is lacking. To overcome these limitations, we engineered a shRNA encoding LentiViral Dual Promoter vector (shLVDP) that enabled real-time monitoring of mesenchymal stem (MSC) differentiation and simultaneous gene knockdown. In this vector, the activity of the alpha-smooth muscle actin (aSMA) promoter was measured by the expression of a destabilized green fluorescent protein, and was used as an indicator of myogenic differentiation; constitutive expression of discosoma red fluorescent protein was used to measure transduction efficiency and to normalize aSMA promoter activity; and shRNA was encoded by a doxycycline (Dox)-regulatable H1 promoter. Importantly, the normalized promoter activity was independent of lentivirus titer allowing quantitative assessment of gene knockdown. Using this vector, we evaluated 11 genes in the TGF-b1 or Rho signaling pathway on SMC maturation and on MSC differentiation along the myogenic lineage. As expected, knockdown of genes such as Smad2/3 or RhoA inhibited myogenic differentiation, while knocking down the myogenic differentiation inhibitor, Klf4, increased aSMA promoter activity significantly. Notably, some genes for example, Smad7 or KLF4 showed differential regulation of myogenic differentiation in MSC from different anatomic locations such as bone marrow and hair follicles. Finally, Dox-regulatable shRNA expression enabled temporal control of gene knockdown and provided dynamic information on the effect of different genes on myogenic phenotype. Our data suggests that shLVDP may be ideal for development of lentiviral microarrays to decipher gene regulatory networks of complex biological processes such as stem cell differentiation or reprogramming.

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Section: Introductionmentioning

confidence: 99%

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

et al. 2012

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“…First, novel self-renewal genes were identified by expression profiling for genes highly expressed in ES cells followed by functional genetic studies (Ivanova et al 2006;Zhang et al 2006). Second, a protein network involved in self-renewal was discovered by biochemical purification of proteins interacting with known pluripotency factors Liang et al 2008).…”

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confidence: 99%

A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal

Hu¹,

Kim²,

Xu³

et al. 2009

Genes Dev.

361

423

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We performed a genome-wide siRNA screen in mouse embryonic stem (ES) cells to identify genes essential for selfrenewal, and found 148 genes whose down-regulation caused differentiation. Many of the identified genes function in gene regulation and/or development, and are highly expressed in ES cells and embryonic tissues. We further identified target genes of two transcription regulators Cnot3 and Trim28. We discovered that Cnot3 and Trim28 co-occupy many putative gene promoters with c-Myc and Zfx, but not other pluripotency-associated transcription factors. They form a unique module in the self-renewal transcription network, separate from the core module formed by Nanog, Oct4, and Sox2. The transcriptional targets of this module are enriched for genes involved in cell cycle, cell death, and cancer. This supports the idea that regulatory networks controlling self-renewal in stem cells may also be active in certain cancers and may represent novel anti-cancer targets. Our screen has implicated over 100 new genes in ES cell self-renewal, and illustrates the power of RNAi and forward genetics for the systematic study of self-renewal.[Keywords: ES cells; self-renewal; RNAi; genetic screen] Supplemental material is available at http://www.genesdev.org.

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“…Oct4, Sox2, and Nanog participate in a transcriptional network with essential functions in the formation and/or maintenance of both the ICM during mouse preimplantation development and murine ES cells in culture [7][8][9][10]. Additional transcription factors have been implicated in stem cell biology and pluripotency, based on specific expression patterns [11][12][13], loss-of-function studies [14,15], or epigenetic contributions [16]. Among those genes activated during ZGA and restrictively expressed in preimplantation embryos is Zscan4 [17], which is expressed in 2-cell stage embryos [18] and a subset of ES cells [19].…”

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confidence: 99%

Functional Analysis ofRex1During Preimplantation Development

Climent

Alonso-Martín

Pérez-Palacios

et al. 2013

Stem Cells and Development

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Rex1/Zfp42 is a nuclear protein that is highly conserved in mammals, and widely used as an embryonic stem (ES) cell marker. Although Rex1 expression is associated with enhanced pluripotency, loss-of-function models recently described do not exhibit major phenotypes, and both preimplantation development and ES cell derivation appear normal in the absence of Rex1. To better understand the functional role of Rex1, we examined the expression and localization of Rex1 during preimplantation development. Our studies indicated that REX1 is expressed at all stages during mouse preimplantation development, with a mixed pattern of nuclear, perinuclear, and cytoplasmic localization. Chromatin association seemed to be altered in 8-cell embryos, and in the blastocyst, we found REX1 localized almost exclusively in the nucleus. A functional role for Rex1 in vivo was assessed by gain-and loss-of-function approaches. Embryos with attenuated levels of Rex1 after injection of zygotes with siRNAs did not exhibit defects in preimplantation development in vitro. In contrast, overexpression of Rex1 interfered with cleavage divisions and with proper blastocyst development, although we failed to detect alterations in the expression of lineage and pluripotency markers. Rex1 gain-and loss-of-function did alter the expression levels of Zscan4, an important regulator of preimplantation development and pluripotency. Our results suggest that Rex1 plays a role during preimplantation development. They are compatible with a role for Rex1 during acquisition of pluripotency in the blastocyst.

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Screening for Genes Essential for Mouse Embryonic Stem Cell Self‐Renewal Using a Subtractive RNA Interference Library

Cited by 42 publications

References 26 publications

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal

Functional Analysis ofRex1During Preimplantation Development

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