Conditional RNAi Using the Lentiviral GLTR System

Pfeiffenberger, Elisabeth; Sigl, Reinhard; Geley, Stephan

doi:10.1007/978-1-4939-3753-0_10

Cited by 5 publications

(5 citation statements)

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“…Cell culture, cloning, and plasmids All cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at 37°C in humidified conditions with 5% CO 2 . U2OS photoactivatable GFP (PA-GFP)-a-tubulin (Ganem et al, 2005) cell line expressing stable inducible short hairpin RNA (shKIF4A) targeting KIF4A sequence 5 0 -GCAAGATCCTGAAAGAGAT-3 0 was generated using a multipurpose GATEWAY-based lentiviral tetracyclineregulated conditional RNAi system (GLTR) using pENTR-THT-III (Addgene plasmid #55791) and pGLTR-X-Puro (Addgene plasmid #58246) plasmids (Sigl et al, 2014;Pfeiffenberger et al, 2016). Stable inducible U2OS PA-GFP-a-tubulin KIF4A shRNA cell lines conditionally expressing RNAi-resistant mCherry-KIF4A variants were generated by lentiviral infection followed by clonal selection with antibiotics.…”

Section: Methodsmentioning

confidence: 99%

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

et al. 2020

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Section: Methodsmentioning

confidence: 99%

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

et al. 2020

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“…All cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at 37°C in humidified conditions with 5% CO2. U2OS photoactivatable GFP (PA-GFP)-α-tubulin (Ganem et al, 2005) cell line expressing stable inducible short hairpin RNA (shKIF4A) targeting KIF4A sequence 5'-GCAAGATCCTGAAAGAGAT-3' was generated using a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR) using pENTR-THT-III (Addgene plasmid #55791) and pGLTR-X-Puro (Addgene plasmid #58246) plasmids (Pfeiffenberger, Sigl et al, 2016, Sigl et al, 2014. Stable inducible U2OS PA-GFP-α-tubulin KIF4A shRNA cell lines conditionally expressing RNAi-resistant mCherry-KIF4A variants were generated by lentiviral infection followed by clonal selection with antibiotics.…”

Section: Cell Culture Cloning and Plasmidsmentioning

confidence: 99%

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

Steblyanko

Rajendraprasad

Osswald³

et al. 2020

Preprint

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Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. We show that both of these activities work in coordination with MTcrosslinking motors kinesin-5/EG5 and kinesin-12/KIF15. Our data further indicate that MT-flux driving force is transmitted from non-KT MTs to KT-MTs via MT-coupling by HSET and NuMA.Moreover, we found that MT-flux rate correlates with spindle size and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we revealed that flux is required to counteract the kinesin 13/MCAK-dependent MT-depolymerization to regulate spindle length.Thus, our study demonstrates that MT-flux in human cells is driven by the coordinated action of four kinesins, and is required to regulate mitotic spindle size in response to MCAK-mediated MTdepolymerizing activity at KTs. Keywords Kinesins / Kinetochore / Microtubules / Mitosis / Mitotic spindleWe thank Duane Compton and Rene Medema for providing the U2OS PA-GFP-tubulin and U2OS PA-GFP-tubulin/mCherry-tubulin cell lines, respectively. We thank Claire Walczak for providing the GFP-HSET and GFP-HSET N593K plasmids. We thank Martina Barisic for technical assistance. Author contributions YS, GR, AJP, HM and MB designed experiments; YS generated tools and performed and analyzed most of the experiments; YS and MB performed photoactivation experiments, image quantification and analysis. GR performed and analyzed the spindle size-related experiments. MO performed and analyzed initial photoactivation experiments; YS, AJP and HM performed CH-STED experiments and analysis; SG provided reagents; SE contributed to designing and analyzing the experiments; MB, YS and GR wrote the manuscript, with contributions from all authors; MB conceived and coordinated the project.

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“…Currently, siRNA import into cells requires vectors such as plasmids and recombinant lentiviruses that express shRNAs efficiently and stably. As recombinant lentiviruses have a higher adaptability and replication ability in host cells, and as their genes can be integrated into cellular genomes, the silencing effect of lentiviruses is more efficient than plasmids [30]. However, this kind of gene integration may induce host genome mutations and cause cellular injury.…”

Section: Discussionmentioning

confidence: 99%

Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector

Wang

Shi

et al. 2019

Vaccines

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Porcine epidemic diarrhea (PED) re-emerged in China in 2010 and is now widespread. Evidence indicates that highly virulent porcine epidemic diarrhea virus (PEDV) strains belonging to genotype G2 caused a large-scale outbreak of diarrhea. Currently, vaccines derived from PEDV classical strains do not effectively prevent infection by virulent PEDV strains, and no specific drug is available to treat the disease. RNA interference (RNAi) is a novel and effective way to cure a wide range of viruses. We constructed three short hairpin RNA (shRNA)-expressing plasmids (shR-N307, shR-N463, and shR-N1071) directed against nucleocapsid (N) and determined their antiviral activities in intestine epithelial cells infected with a classical CV777 strain and LNCT2. We verified that shR-N307, shR-N463, and shR-N1071 effectively inhibited the expression of the transfected N gene in vitro, comparable to the control shRNA. We further demonstrated the shRNAs markedly reduced PEDV CV777 and LNCT2 replication upon downregulation of N production. Therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors.Vaccines 2019, 7, 173 2 of 12 the CV777 vaccine strain belonging to subtype G1 [11]. However, many studies demonstrated that commercially available PEDV vaccines derived from classical strains of PEDV do not provide effective protection against highly virulent PEDV variant infections in China [3]. Thus, PEDV infection remains a major veterinary problem. Understanding it is essential to developing novel antiviral drugs that will specifically inhibit PEDV propagation.RNA interference (RNAi) is a short, double-strand RNA-induced process that targets and degrades the messenger RNA (mRNA) of specific sequences [12,13]. Post-transcriptional gene silencing can be mediated by exogenous small interfering RNAs (siRNAs), endogenous microRNAs (miRNAs), and short hairpin RNAs (shRNAs). Effective gene knockdown is achieved by shRNAs by inducing the endogenous RNAi process [14,15]. Transcribed shRNAs are exported from the nucleus by Exportin-5 and processed by the RNase III Dicer into small double-stranded RNA (dsRNA) molecules of 19 to 23 bp called siRNAs. The complementary guide strand of processed siRNA is incorporated into the RNA-induced silencing complex to mediate the cleavage of target mRNAs [16,17]. RNAi evolves in the host defense system directed at infectious viruses and transposable elements. Effective silencing of transgene expression and endogenous genes in vivo was shown by a number of groups [6,7,18]. These findings raised the possibility that RNAi could be another therapeutic approach for inhibiting virus infection. ShRNAs were employed for therapy against human viral diseases, as well as cancer and neurogenerative diseases [8]. PEDV primarily infects villous epithelial cells throughout the small intestine and causes serious injury of intestine epithelial cells (IECs), including superficial villous enterocyte swelling and severe diffuse atrophic enterit...

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Conditional RNAi Using the Lentiviral GLTR System

Cited by 5 publications

References 18 publications

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector

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