Constitutive and Inducible Systems for Genetic &lt;em&gt;In Vivo &lt;/em&gt;Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hubner, Eric K.; Lechler, Christian; Rosner, Thomas; Kohnke-Ertel, Birgit; Schmid, Roland M.; Ehmer, Ursula

doi:10.3791/56613

Cited by 6 publications

(7 citation statements)

References 43 publications

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“…In this regard, it is of note that we succeeded in inducing targeted DNA demethylation of the Fgf21 promoter using the dCas9-SunTag and scFv-TET1CD system in PPARα-KO mouse livers using HTVi. It has been reported that the transfection efficiency of HTVi in mouse liver is 5-40% [30][31][32] , indicating that the transfection efficiency (about 4%) with pPlatTET-gRNA2 all-in-one vector was less than expected. However, the transfection efficiency of HTVi in mouse liver mainly depended on the size of the injected construct.…”

Section: Discussionmentioning

confidence: 88%

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Hanzawa

Hashimoto

Yuan

et al. 2020

Sci Rep

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Recently, we reported ppARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DnA methylation is associated with enhanced gene expression after ppARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DnA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DnA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to ppARα synthetic ligand administration and fasting, respectively. this study provides direct evidence that the DnA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues. In mammalian cells, DNA methylation is a major epigenetic modification, which regulates gene expression without alteration of the DNA sequence and thus plays a pivotal role in a myriad of physiological and pathological processes, including cell development and differentiation, genome imprinting, and tumorigenesis 1. We reported previously that the DNA methylation status of hepatic metabolic genes dynamically changes in early life, especially during the suckling period, thereby sequentially developing metabolic function in the liver to adapt to the drastic changes in the major nutrition source 2-4. Peroxisome proliferator-activated receptor-α (PPARα) is a nuclear receptor and a key regulator of hepatic lipid metabolism, which is activated by milk lipids as ligands at the onset of lactation. PPARα governs the transcription of major hepatic metabolism-related genes, and the activation of PPARα physiologically leads to DNA demethylation of fatty acid β-oxidation genes in the postnatal mouse liver 3,4. Of note, administration of a synthetic PPARα ligand to mouse dams during the perinatal period induced enhanced reduction of DNA methylation of PPARα target genes in the offspring liver, suggesting that the DNA methylation status of PPARα target genes can be modulated with ease during the perinatal period 3,4. A genome-wide analysis of DNA methylation revealed that a few PPARα target genes undergo ligand-activated, PPARα-dependent DNA demethylation during the perinatal period, and the DNA hypomethylation status of these persists into adulthood. Among these genes, which may be referred to as "epigenetic memory genes," we focused on fibroblast growth factor 21 (FGF21), which is a metabolic hormone derived from the liver and a master regulator of glucose and lipid metabolism 5-7 .

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Section: Discussionmentioning

confidence: 88%

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Hanzawa

Hashimoto

Yuan

et al. 2020

Sci Rep

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“…Minicircle DNA [33,114,124,132,136,137,143,171] US-targeted microbubble destruction [178] PhiC31 Integrase [197] microRNA [47,150,152,[188][189][190][191] Computer-assisted hydrodynamic delivery [12,198] Sleeping Beauty [30,96,111,142,191,[199][200][201][202] Circular RNA [126] Bioluminescence imaging [47,[203][204][205][206] piggyBac [126,207] shRNA [94,192,193] Tissue clearing [206] Cre-loxP [208][209][210] siRNA [87,[194][195][196] Repopulation [189,211] CreER…”

Section: Delivery Materials Technological Developments Gene Editingmentioning

confidence: 99%

“…Aiming at longlasting gene expression using nonviral vectors and in addition to epigenetic control [227], plasmids that replicate in an episomal fashion and site-specific integration systems were developed and hydrodynamically delivered. In terms of site-specific integration, PhiC31 integrase [197], sleeping beauty transposon [30,96,111,142,191,[199][200][201][202], piggyBac transposon [126,207], and Cre-loxP and technologies derived from it [208][209][210] were a major focus. Tamoxifen-dependent Cre recombinase (CreER) can initiate the recombination event at any desired time point [201,212].…”

Section: Delivery Materials Technological Developments Gene Editingmentioning

confidence: 99%

Hydrodynamic Delivery: Characteristics, Applications, and Technological Advances

et al. 2023

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The principle of hydrodynamic delivery was initially used to develop a method for the delivery of plasmids into mouse hepatocytes through tail vein injection and has been expanded for use in the delivery of various biologically active materials to cells in various organs in a variety of animal species through systemic or local injection, resulting in significant advances in new applications and technological development. The development of regional hydrodynamic delivery directly supports successful gene delivery in large animals, including humans. This review summarizes the fundamentals of hydrodynamic delivery and the progress that has been made in its application. Recent progress in this field offers tantalizing prospects for the development of a new generation of technologies for broader application of hydrodynamic delivery.

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“…For the methods of i.v. injection, we performed the experiments as previous described (Hubner et al, 2018). Briefly, the mice were restrained by being placed in a 50 ml conical tube with the tip cut off to allow for adequate air circulation.…”

Section: In Vivo Drug Treatmentmentioning

confidence: 99%

Quercetin relieves D‐amphetamine‐induced manic‐like behaviour through activating TREK‐1 potassium channels in mice

Ren

Liu

Guo

et al. 2021

British J Pharmacology

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Background and Purpose: Quercetin is a well-known plant flavonoid with neuroprotective properties. Earlier work suggested it may relieve psychiatric disorders, cognition deficits and memory dysfunction through anti-oxidant and/or radical scavenging mechanisms. In addition, quercetin modulated the physiological function of some ion channels. However, the detailed ionic mechanisms of the bioeffects of quercetin remain unknown. Experimental Approach: Effects of quercetin on neuronal activities in the prefrontal cortex (PFC) and its ionic mechanisms were analysed by calcium imaging using mice bearing a green fluorescent protein, calmodulin, and M13 fusion protein and patch clamp in acute brain slices from C57BL/6 J mice and in HEK 293 cells. The possible ionic mechanism of action of quercetin on D-amphetamine-induced manic-like effects in mice was explored with c-fos staining and the open field behaviour test.Key Results: Quercetin reduced calcium influx triggered by PFC pyramidal neuronal activity. This effect involved increasing the rheobase of neuronal firing through decreasing membrane resistance following quercetin treatment. Spadin, a blocker of TREK-1 potassium channels, also blocked the effect of quercetin on the membrane resistance and neuronal firing. Further, spadin blocked the neuroprotective effects of quercetin. The effects of quercetin on TREK-1 channels could be mimicked by GF109203X, a protein kinase C inhibitor. In vivo, injection of quercetin relieved the manic hyperlocomotion in mice, induced by D-amphetamine. This action was partly alleviated by spadin. Conclusion and Implications:TREK-1 channels are a novel target for quercetin, by inhibiting PKC. This action could contribute to both the neuroprotective and antimanic-like effects.

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Constitutive and Inducible Systems for Genetic <em>In Vivo </em>Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Cited by 6 publications

References 43 publications

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Hydrodynamic Delivery: Characteristics, Applications, and Technological Advances

Quercetin relieves D‐amphetamine‐induced manic‐like behaviour through activating TREK‐1 potassium channels in mice

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