[36] Generation of a destabilized form of enhanced green fluorescent protein

Zhao, Xiaoning; Jiang, Xin; Huang, Chiao-Chian; Kain, Steven R.; Li, Xianqiang

doi:10.1016/s0076-6879(99)02038-8

Cited by 7 publications

(6 citation statements)

References 7 publications

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“…A comparison between GFP expression in cycling mice those using uteri recovered after induction of endometrial shedding/repair led us to conclude that the presence of GFP in the epithelial cells was most likely because the turnover of the GFP was slow enough for it to persist after the Pgdfrb promoter was no longer active. This would be consistent with reports that eGFP protein was engineered to have increased fluorescence and slower turnover in vivo (Zhao et al, 1999); although the half-life of the protein varies in different cell models a paper exploring in vivo expression in cancer reported it to be ~15h (Danhier et al, 2015). These results were also considered consistent with detection of less GFP in EPCAM+ epithelial cells at 48 than 24h when this was evaluated using flow cytometry.…”

Section: Discussionsupporting

confidence: 88%

Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

Kirkwood

Gibson

Shaw

et al. 2021

Preprint

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The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study we used a validated mouse model of endometrial repair in combination with three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the cycling endometrium. Using scRNAseq we identified a novel population of PDGFRb+ cells that arose in the endometrium in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they arose from stromal fibroblasts and were in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2+). We demonstrate for the first time that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to restoration of tissue integrity during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Ashermans syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding.

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Section: Discussionsupporting

confidence: 88%

Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

Kirkwood

Gibson

Shaw

et al. 2021

Preprint

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“…Obviously, there is a conflict between the normal twdlM ‐promoter‐driven GFP expression and the low level of endogenous twdlM expression detected by quantitative polymerase chain reaction (qPCR). We reckon that the persistence of the GFP protein or derivatives like RFP of around 26 hr (Corish & Tyler‐Smith, ; Li et al, ; Zhao, Jiang, Huang, Kain, & Li, ) expressed before inhibitor feeding masks the downregulation of the promoter activity upon feeding. At Day 2, by contrast, RFP or GFP expression is markedly reduced in the inhibitor‐fed larvae.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of fatty acid desaturation impairs cuticle differentiation in Drosophila melanogaster

Wang

Maier

Gehring

et al. 2019

Arch Insect Biochem Physiol

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Previously, we showed that inhibition of the activity of fatty acid desaturases (Desat) perturbs signalling of the developmental timing hormone ecdysone in the fruit fly Drosophila melanogaster. To understand the impact of this effect on cuticle differentiation, a process regulated by ecdysone, we analysed the cuticle of D. melanogaster larvae fed with the Desat inhibitor CA10556. In these larvae, the expression of most of the key cuticle genes is normal or slightly elevated at day one of CA10556 feeding. As an exception, expression of twdlM coding for a yet uncharacterised cuticle protein is completely suppressed. The cuticle of these larvae appears to be normal at the morphological level. However, these animals are sensitive to desiccation, a trait that according to our data, among others, may be associated with reduced TwdlM amounts. At day two of CA10556 feeding, expression of most of the cuticle genes tested including twdlM is suppressed. Expression of cpr47Eb coding for a chitinbinding protein is, by contrast, highly elevated suggesting that Cpr47Eb participates at a specific compensation program. Overall, the cuticle of these larvae is thinner than the cuticle of control larvae. Taken together, lipid desaturation is necessary for a coordinated deployment of a normal cuticle differentiation program. K E Y W O R D S barrier, cuticle, differentiation, epidermis, lipids Arch. Insect Biochem. Physiol. 2019;100:e21535.wileyonlinelibrary.com/journal/arch

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“…We transfected siRNA expression vectors into RV_HSCR‐c cells and monitored both the expression level of the EGFP mRNA and the production of the viruses to determine the effect of siRNA on the virus replication after viral integration. EGFP has an estimated half‐life of >24 h in vivo (50). It appears to be stable when expressed in mammalian cells.…”

Section: Resultsmentioning

confidence: 99%

Potent and Specific Inhibition of Retrovirus Production by Coexpression of Multiple siRNAs Directed Against Different Regions of Viral Genomes

Jin

2006

Biotechnol. Prog.

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siRNA-mediated RNA degradation has been demonstrated to act as an antiviral system in many species. Here we describe inhibition of retrovirus production by multiple siRNAs designed to target various regions of the viral genomes. Using murine leukemia virus (MuLV) as a model, we demonstrate that the virus production can be inhibited by 77% in siLTR2 (a siRNA targeting the U3 region of MuLV) expression vector transfected cells. Coexpression of siLTR2 with siPsi2 (a siRNA targeting the 3' Psi (packaging signal sequence) results in 93% suppression of the virus production, suggesting that an increased inhibition of the virus production can be achieved by coexpression of multiple siRNAs to target different regions of the viral RNA simultaneously. Our results also indicate that not all sequences of the viral RNA are equally accessible to siRNA. We show that U3 region of MuLV is more accessible to siRNA, whereas the packaging signal sequence, especially the region adjacent to 5'LTR, is less accessible to siRNA, partly as a result of the binding of Gag precursors. Furthermore, we demonstrate that coexpression of siLTR2 with siPsi2 in virus producer cells leads to 88% knockdown of viral titer, showing the benefit of coexpression of multiple siRNAs for potent suppression of virus production in the setting of an established infection. Moreover, we demonstrate that infection of MuLV in cells that stably coexpress siLTR2 with siPsi2 diminishes by 77%. Taken together, we establish that siRNA-mediated gene silencing can suppress multiple steps of the retrovirus life cycle, offering a potential for both treating virus-associated diseases and preventing viral infection.

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[36] Generation of a destabilized form of enhanced green fluorescent protein

Cited by 7 publications

References 7 publications

Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

Inhibition of fatty acid desaturation impairs cuticle differentiation in Drosophila melanogaster

Potent and Specific Inhibition of Retrovirus Production by Coexpression of Multiple siRNAs Directed Against Different Regions of Viral Genomes

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