Generation of a New Gateway-Compatible Inducible Lentiviral Vector Platform Allowing Easy Derivation of Co-Transduced Cells

Groote, Philippe De; Grootjans, Sasker; Lippens, Saskia; Eichperger, Chantal; Leurs, Kirsten; Kahr, Irene; Tanghe, Giel; Bruggeman, Inge; Schamphelaire, Wouter De; Urwyler, Corinne; Vandenabeele, Peter; Haustraete, Jurgen; Declercq, Wim

doi:10.2144/000114417

Cited by 12 publications

(9 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The sequence was cloned into pENTR3C using the CloneEZ PCR Cloning Kit (GenScript, Piscataway, NJ, USA), and substitution of the Asparagine residues into Glutamine residues was achieved by QuikChange® Site-Directed Mutagenesis Kit (Agilent, Santa Clara, US). Sequences were then transferred into pcDNA3, and custom-made doxycyline-inducible pDG2(blast)-rtTA3-FLAG lentiviral destination vectors 46 using the LR Gateway recombination system (Life Technologies, Carlsbad, CA, USA). Transient overexpression of mTRAIL-R mutants in Trail-R − / − MEFs was done by transfection of pcDNA3 plasmids using JetPRIME® transfection reagent (Polyplus-transfection® SA, Illkirch, France), following the manufacturer’s recommendations.…”

Section: Methodsmentioning

confidence: 99%

N-glycosylation of mouse TRAIL-R restrains TRAIL-induced apoptosis

et al. 2018

Self Cite

View full text Add to dashboard Cite

The sensitivity of cells to death receptor-induced apoptosis is commonly controlled by multiple checkpoints in order to limit induction of excessive or unnecessary death. Although cytotoxic in various cancer cells, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) does not trigger apoptosis in most non-transformed cells. The molecular nature of the checkpoints that normally protect the cells from TRAIL-induced death are not fully understood. Endoplasmic reticulum (ER) stress has been reported to switch the sensitivity of human cells to the cytotoxic effect of TRAIL, suggesting that this cellular state perturbs some of these protective mechanisms. We found that tunicamycin (TU), but no other ER stress inducers, sensitized mouse fibroblasts and hippocampal neuronal cells to TRAIL-induced apoptosis. Importantly, the sensitization was specific to TRAIL and not caused by differences in ER stress induction. Instead, it relied on the inhibition of N-glycosylation of the mouse TRAIL receptor (mTRAIL-R). Inhibition of N-glycosylation did not alter cell surface expression of mTRAIL-R but enhanced its ability to bind TRAIL, and facilitated mTRAIL-R oligomerization, which resulted in enhanced death-inducing signaling complex (DISC) formation and caspase-8 activation. Remarkably, reconstitution of mTRAIL-R-deficient cells with a version of mTRAIL-R mutated for the three N-glycosylation sites identified in its ectodomain confirmed higher sensitivity to TRAIL-induced apoptosis. Together, our results demonstrate that inhibition of N-glycosylation of mTRAIL-R, and not ER stress induction, sensitizes mouse cells to TRAIL-induced apoptosis. We therefore reveal a new mechanism restraining TRAIL cytotoxicity in mouse cells.

show abstract

Section: Methodsmentioning

confidence: 99%

N-glycosylation of mouse TRAIL-R restrains TRAIL-induced apoptosis

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…Since multiple different GFP variants are used to generate reporter RNA viruses, we sequenced four plasmids that encode other GFP variants ( Supplementary Figure S2 ) 23 24 . A drop in coverage at ‘CCNGCC’ or the shorter ‘CNGCC’ motif can also be observed in some of the sequences encoding these GFP variants 23 24 . Although the ‘CCNGCC’ motif is absent in the Aequorea victoria GFP coding sequence, the shorter ‘CNGCC’ motif occurs two times in this sequence (positions 820 to 824 and 975 to 979, plasmid numbering).…”

Section: Resultsmentioning

confidence: 99%

“…The sequence of NS1(1-73)Dmd-GFP-NEP and the introduced C1398T or/and C1401T mutations were confirmed by Sanger sequencing on a capillary sequencer (Applied Biosystems 3730XL DNA Analyzer). Plasmids pBluAGFP 24 , pEF6-turboGFP-MCS, pLVX-EF1a-IRES-ZsGreen1 (Clontech-BD Biosciences, Palo Alto, United States) and pDG2-hRIPK4-WT-EGFP-puro 23 were kindly provided by the BCCM/LMBP Plasmid Collection, Dr. Jens Staal and Giel Tanghe from our Department.…”

Section: Methodsmentioning

confidence: 99%

Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene

Hoecke

Verhelst

Saelens

2016

Sci Rep

View full text Add to dashboard Cite

Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present when virus-derived RT-PCR product or the parental plasmid DNA was used as starting material for NGS and regardless of whether Nextera XT transposase or Covaris shearing was used for DNA fragmentation. Therefore, the sequence coverage dip in the GFP coding sequence was not the result of emerging GFP mutant viruses or a bias introduced by Nextera XT fragmentation. Instead, we found that the Illumina MiSeq sequencing method disfavours the ‘CCCGCC’ motif in the GFP coding sequence.

show abstract

“…When using a double antibiotic approach, the mechanism of action of both antibiotics should not interfere with the interaction of DNA or RNA processes that may inhibit CRISPR/Cas9 activity. Both antibiotics must also be compatible and not show any signs of drug-drug interaction – a condition that is met by the combination of puromycin and blasticidin used in this study 15–17 .…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection

Supharattanasitthi

Carlsson

Sharif

et al. 2019

Sci Rep

View full text Add to dashboard Cite

CRISPR/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes. The donor templates were co-transfected alongside the CRISPR/Cas9 machinery into cells and a double antibiotic selection was optimised and allowed the isolation of viable desired clones. We applied the method for obtaining isogenic cells homozygous for variant B cystatin C, a recessive risk factor for age-related macular degeneration and Alzheimer’s disease, in both induced Pluripotent Stem Cells (iPSCs) and a human RPE cell line. Bi-allelic gene edited clones were validated by sequencing, demonstrating that the double antibiotic templates approach worked efficiently for both iPSCs and human differentiated cells. We propose that this one step gene editing approach can be used to improve the specificity and frequency of introducing homozygous modifications in mammalian cells.

show abstract

Generation of a New Gateway-Compatible Inducible Lentiviral Vector Platform Allowing Easy Derivation of Co-Transduced Cells

Cited by 12 publications

References 22 publications

N-glycosylation of mouse TRAIL-R restrains TRAIL-induced apoptosis

N-glycosylation of mouse TRAIL-R restrains TRAIL-induced apoptosis

Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene

CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection

Contact Info

Product

Resources

About