Influence of <i>lox</i>P insertion upstream of the <i>cis</i>‐acting packaging domain on adenovirus packaging efficiency

Kanegae, Yumi; Ishimura, Masakazu; Kondo, Shu; Saito, Izumu

doi:10.1111/j.1348-0421.2012.00454.x

Cited by 3 publications

(2 citation statements)

References 39 publications

(87 reference statements)

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“…For knockdown experiments, HepG2 cells were transfected with 25 nM SMARTpool siRNA for ATG7 , p62 , or NCoR1 using Dharmafect 1 (Thermo Fisher Scientific, Waltham, MA, USA). NCoR1 was expressed using a helper-dependent adenovirus vector system 62 containing loxP at position 143 63 . To exogenously express GFP, ATG7, or ATG7 C572S , we used the Adenovirus Expression Vector Kit (Takara Bio, Kusatsu, Japan).…”

Section: Methodsmentioning

confidence: 99%

Autophagy regulates lipid metabolism through selective turnover of NCoR1

et al. 2019

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Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in β-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates β-oxidation and ketone bodies production.

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Section: Methodsmentioning

confidence: 99%

Autophagy regulates lipid metabolism through selective turnover of NCoR1

et al. 2019

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“…14 Moreover, tweaking the HV packaging domain can delay its packaging, 15 or reduce its packaging efficiency in co-infection contexts. 16,17 Reduced HDV production obtained after the initial transfection step, limited by transfection efficiency and initiation of viral DNA replication from plasmid substrates, has also prompted others to improve the strategies. With a focus on this step, production of HDV was improved by creating cell lines expressing pre-terminal protein and adenoviral DNA polymerase, 18 optimizing transfection methods compliant with scalable processes 9 and developing an intracellular system to release HDV genome from transfected circular plasmids, which increased transfection efficiencies of 10-fold.…”

Section: Introductionmentioning

confidence: 99%

Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors

et al. 2014

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Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction.

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Activity-regulated micro-exon splicing programs underlie late-onset plasticity at the axon initial segment

Darwish,

Suzuki,

Ogawa

et al. 2023

Preprint

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The axon initial segment (AIS) is a specialized neuronal compartment located at the proximal end of axons and initiates action potentials. AIS undergoes plastic changes with aging, disease, and activity levels; however, the molecular mechanisms underlying their plasticity remain unclear. We discovered that depolarization induces diffuse elongation of the AIS in cerebellar granule cells over the span of days via the Ca2+-dependent ERK/MAP kinase pathway. These structural changes were accompanied by a decrease in voltage-gated Na+channel density, resulting in a homeostatic attenuation in neuronal excitability. Notably, we found that the late-onset AIS plasticity is associated with depolarization-induced alternative splicing of smaller exons (<100 nt) of transcripts encoding AIS-enriched proteins. In addition, depolarization-induced the skipping of the 53-nt exon19 from the transcript of the splicing protein Rbfox1. CRISPR-mediated removal of exon 19 from Rbfox1 promoted its nuclear localization and sequentially induced a series of downstream micro-exon splicing changes in several AIS proteins, recapitulating cerebellar AIS plasticity. In a Rbfox1-independent mechanism, depolarization-induced insertion of the developmentally regulated micro-exon 34 into the key AIS scaffolding protein Ankyrin G (AnkG). The constitutive insertion of exon 34 into AnkG disrupted its interaction with the AIS cytoskeletal protein βIV spectrin and induced plastic changes in the AIS. Our findings provide fundamental mechanistic insights into the activity-mediated late-onset plasticity of AIS, highlighting the power of micro-scale splicing events in the homeostatic regulation of axonal remodeling.

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Influence of loxP insertion upstream of the cis‐acting packaging domain on adenovirus packaging efficiency

Cited by 3 publications

References 39 publications

Autophagy regulates lipid metabolism through selective turnover of NCoR1

Autophagy regulates lipid metabolism through selective turnover of NCoR1

Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors

Activity-regulated micro-exon splicing programs underlie late-onset plasticity at the axon initial segment

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