A Cassette System to Study Embryonic Stem Cell Differentiation by Inducible RNA Interference

Wegmüller, Daniel; Raineri, Ines; Gross, B.; Oakeley, Edward J.; Moroni, Christoph

doi:10.1634/stemcells.2006-0106

Cited by 12 publications

(8 citation statements)

References 41 publications

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“…For example, a previous study showed that siRNA inhibition of ZFP36L1 expression in mouse myoblasts inhibits cell differentiation 58. However, a different investigation showed that induction of shRNA specific for ZFP36L1 strongly stimulates cardiomyocyte formation within embryoid bodies 59. In contrast, ZFP36L2 knockdown promoted adipogenesis compared with the shRNA control, indicating that TTP family proteins might target different mRNAs and play different functional roles under physiological conditions.…”

Section: Discussionmentioning

confidence: 99%

Differential Expression and Functional Analysis of the Tristetraprolin Family during Early Differentiation of 3T3-L1 Preadipocytes

Lin¹,

Lin²,

Yang³

et al. 2012

Int. J. Biol. Sci.

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The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3′ untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3′UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.

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Section: Discussionmentioning

confidence: 99%

Differential Expression and Functional Analysis of the Tristetraprolin Family during Early Differentiation of 3T3-L1 Preadipocytes

Lin¹,

Lin²,

Yang³

et al. 2012

Int. J. Biol. Sci.

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“…When ectopically expressed TIS11b promoted myeloid progenitor L-G cell proliferation and inhibited differentiation [62]. Intriguingly, siRNA mediated TIS11b knockdown in murine embryonic stem cells promoted their differentiation to cardiomyocytes [106]. Increased expression of TIS11b was reported in primary breast tumours [107] and hepatocellular carcinomas [108].…”

Section: Tis11 Family In Cell Functions and Diseasementioning

confidence: 99%

TIS11 Family Proteins and Their Roles in Posttranscriptional Gene Regulation

Baou

Jewell

Murphy

2009

BioMed Research International

103

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Posttranscriptional regulation of gene expression of mRNAs containing adenine-uridine rich elements (AREs) in their 3′ untranslated regions is mediated by a number of different proteins that interact with these elements to either stabilise or destabilise them. The present review concerns the TPA-inducible sequence 11 (TIS11) protein family, a small family of proteins, that appears to interact with ARE-containing mRNAs and promote their degradation. This family of proteins has been extensively studied in the past decade. Studies have focussed on determining their biochemical functions, identifying their target mRNAs, and determining their roles in cell functions and diseases.

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“…Inducible RNAi raises the prospect of temporal and dosage-sensitive control over the induction of gene knockdown. Previously reported strategies for inducible RNAi in ES cells or transgenic mice include the use of tamoxifen-inducible Cre-recombinase to activate vector-based shRNAs and shRNAs integrated in a defined locus (10,11), as well as the use of a doxycyclineinducible shRNA cassette integrated in a defined locus (12,13). Here we describe an alternative drug-inducible system based on the Ainv15 ES cell line, in which an shRNA-mir cassette is directed to the constitutively active hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus upon Cre-mediated sitespecific recombination.…”

Section: Rnai Is a Powerful Tool For Interrogating Gene Function In Ementioning

confidence: 99%

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

Wang

Theunissen

Orkin

2007

Proc. Natl. Acad. Sci. U.S.A.

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RNAi is a powerful tool for interrogating gene function in ES cells.Combining the high penetrance of a microRNA-embedded shRNA (shRNA-mir) cassette with a locus-defined, inducible expression strategy, we developed a system for RNAi in mouse ES cells. An shRNA-mir cassette is targeted near the constitutively active HPRT locus under a tetracycline (tet)-regulatable promoter through Cremediated site-specific recombination. The major advantage of this system is that the shRNA-mir cassette can be targeted to a precise locus, allowing for control of shRNA-mir expression in an inducible fashion. Induction of an shRNA-mir directed against the pluripotency factor, Nanog, resulted in the loss of self-renewal and differentiation to parietal endoderm-like cells, which can be rescued by the introduction of an RNAi-immune version of Nanog cDNA. Knockdown efficiency can be enhanced by using multiple shRNA-mir hairpins against the target gene, which was further validated by knocking down two additional ES cell factors. This site-directed, virus-free, and tet-inducible RNAi system, designated as SDVFi RNAi in our study, presents an efficient option for controlled gene silencing in ES cells.microRNAs ͉ Nanog ͉ RNA interference ͉ tetracycline

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A Cassette System to Study Embryonic Stem Cell Differentiation by Inducible RNA Interference

Cited by 12 publications

References 41 publications

Differential Expression and Functional Analysis of the Tristetraprolin Family during Early Differentiation of 3T3-L1 Preadipocytes

Differential Expression and Functional Analysis of the Tristetraprolin Family during Early Differentiation of 3T3-L1 Preadipocytes

TIS11 Family Proteins and Their Roles in Posttranscriptional Gene Regulation

Site-directed, virus-free, and inducible RNAi in embryonic stem cells

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