Probing tumor phenotypes using stable and regulated synthetic microRNA precursors

Dickins, Ross A.; Hemann, Michael T.; Zilfou, Jack T.; Simpson, David; Ibarra, Ingrid; Hannon, Gregory J.; Lowe, Scott W.

doi:10.1038/ng1651

Cited by 478 publications

(457 citation statements)

References 32 publications

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“…43 This system enables temporal control of shRNA expression at preferred times during the experiment, which is essential for studying genes that may have adverse effect on cell viability or other biological processes, for example, cytoskeletal re-organization. 44 For example, during myogenic differentiation, we observed a considerable level of cytotoxicity in cells actively expressing Rock1 shRNA (data not shown), making data interpretation difficult. Use of the inducible shLVDP enabled TGF-b1-induced differentiation before shRNA expression, thereby allowing us to assess the effect of Rock1 on aSMA promoter activity instead of viability.…”

Section: Discussionmentioning

confidence: 89%

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: Discussionmentioning

confidence: 89%

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

et al. 2012

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“…The two Tnfaip8 shRNAs were then cloned into a modified retroviral expression vector designated pMSCV-LTRmiR30-PIhCD (Supplementary Figure S1b), a modified form of a previously reported vector that embeds the shRNA sequence within a larger miRNA sequence so as to improve effectiveness. 25 To determine the requirement for Tnfaip8 expression for glucocorticoid-mediated thymocyte apoptosis, HPCs were infected with shRNAmiR-expressing retroviruses and used to repopulate 2dGuo depleted FTOCs. The FTOCs were cultured for 13 days and then analyzed for the effect of the target gene knockdown compared with gene overexpression on thymocyte development.…”

Section: Resultsmentioning

confidence: 99%

Tnfaip8 is an essential gene for the regulation of glucocorticoid-mediated apoptosis of thymocytes

et al. 2009

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Glucocorticoids have significant immunoregulatory actions on thymocytes and T cells and act by binding and activating cytosolic glucocorticoid receptors, which translocate to the nucleus and control gene expression through binding to specific response elements in target genes. Glucocorticoids promote cell death by activating an apoptotic program that requires transcriptional regulation. We set out to identify genes that are crucial to the process of glucocorticoid-mediated thymocyte apoptosis. Freshly isolated murine primary thymocytes were treated with dexamethasone, mRNA isolated and used to screen DNA microarrays. A set of candidate genes with upregulated expression was identified and selected members assayed in reconstituted fetal thymic organ culture (FTOC). Fetal liver-derived hematopoietic progenitor cells (HPCs) were infected with retroviruses expressing individual genes then used to repopulate depleted fetal thymic lobes. Reconstituted FTOCs expressing the gene Tnfaip8 were treated with dexamethasone and shown to be greatly sensitized to dexamethasone. Retrovirus-mediated RNA interference was applied to knock down Tnfaip8 expression in HPCs and these were used to reconstitute FTOCs. We observed that downregulating the expression of Tnfaip8 alone was sufficient to effectively protect thymocytes against glucocorticoid-induced apoptosis. We propose that Tnfaip8 is crucial in regulating glucocorticoid-mediated apoptosis of thymocytes.

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“…For example, loss of a single Bim allele or incomplete RNAi-mediated suppression of Puma is sufficient to promote Myc-induced lymphomagenesis. 55,69 Thus, studies that exclusively score the presence or absence of these genes in human tumors may significantly underestimate their relevance to cancer development.…”

Section: Relevance Of the P53-bcl-2 Pathway To Cancermentioning

confidence: 99%

The p53–Bcl-2 connection

Hemann

Lowe²

2006

Cell Death Differ

300

219

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Probing tumor phenotypes using stable and regulated synthetic microRNA precursors

Cited by 478 publications

References 32 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

Tnfaip8 is an essential gene for the regulation of glucocorticoid-mediated apoptosis of thymocytes

The p53–Bcl-2 connection

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