Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization DOI Creative Commons
Arata Honda,

Ryoma Tachibana,

Kazuya Hamada

et al.

Scientific Reports, Journal Year: 2019, Volume and Issue: 9(1)

Published: Aug. 9, 2019

Abstract Rats are effective model animals and have contributed to the development of human medicine basic research. However, application reproductive engineering techniques rats is not as advanced compared with mice, genome editing in has been achieved using embryos obtained by vitro fertilization (IVF). In this study, we conducted superovulation, IVF, knock out IVF rat embryos. We found that superovulation effectively occurred synchronized oestrus cycle anti-inhibin antiserum treatment immature rats, including Brown Norway rat, which a very difficult strain superovulate. Next, collected superovulated oocytes under anaesthesia, offspring derived from were all strains examined. When tyrosinase gene was targeted electroporation these embryos, both alleles disrupted 100% efficiency. Furthermore, long DNA fragment adeno-associated virus knock-in litter high efficiency (33.3–47.4%). Thus, developed methods allow simple efficient production rats.

Language: Английский

Population management using gene drive: molecular design, models of spread dynamics and assessment of ecological risks DOI Creative Commons
Nicolas O. Rode, Arnaud Estoup, Denis Bourguet

et al.

Conservation Genetics, Journal Year: 2019, Volume and Issue: 20(4), P. 671 - 690

Published: April 1, 2019

CRISPR gene drive has recently been proposed as a promising technology for population management, including in conservation genetics. The technique would consist releasing genetically engineered individuals that are designed to rapidly propagate desired mutation or transgene into wild populations. Potential applications biology include the control of invasive pest populations threaten biodiversity (eradication and suppression drives), introduction beneficial mutations endangered (rescue drives). propagation is affected by different factors depend on construct (e.g. its fitness effect timing expression) target species mating system structure). We review potential types drives conservation. examine challenges posed evolution resistance various molecular environmental risks associated with non unintended detrimental ecosystem impacts). provide some guidelines future research discuss ethical, biosafety regulation issues.

Language: Английский

Citations

144
CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection DOI Creative Commons
Sean Chen,

Sabrina Sun,

Dewi Moonen

et al.

Cell Reports, Journal Year: 2019, Volume and Issue: 27(13), P. 3780 - 3789.e4

Published: June 1, 2019

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and replacement; however, existing methods to generate such animals remain laborious costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR delivery with Cas9/sgRNA engineer in the genome high efficiency throughput. We successfully targeted 774 bp fluorescent reporter, 2.1 kb CreERT2 driver, 3.3 expression cassette into loci both embryos live mice. is applicable most widely used knockin schemes requiring lengths within 4.9 packaging capacity. Altogether, efficient, high-throughput, microinjection-free sophisticated engineering potential other mammalian species.

Language: Английский

Citations

95
An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes DOI Creative Commons
Simon E. Tröder, Lena K. Ebert, Linus Butt

et al.

PLoS ONE, Journal Year: 2018, Volume and Issue: 13(5), P. e0196891 - e0196891

Published: May 3, 2018

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols electroporation either require investment specialized electroporators or corrosive pre-treatment which compromises embryo viability. Here, we describe an easily adaptable approach introduction specific mutations C57BL/6 mice by intact using common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison conventional demonstrates significantly reduced physical damage thus improved development successful up 100% living offspring. Hence, our novel Easy Zygotes (EEZy) allows highly efficient generation transgenic while reducing numbers animals required.

Language: Английский

Citations

92
CLICK: one-step generation of conditional knockout mice DOI Creative Commons
Yoshiki Miyasaka, Yoshihiro Uno, Kazuto Yoshimi

et al.

BMC Genomics, Journal Year: 2018, Volume and Issue: 19(1)

Published: May 2, 2018

CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive.Here, we show that electroporation Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), quick generation floxed mice rats.The high efficiency provides homozygous knock-ins oocytes carrying tissue-specific Cre, which allows one-step knockouts founder (F0) mice.

Language: Английский

Citations

89
Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats DOI Creative Commons
Susanne Bäck, Julie Necarsulmer,

Leslie R. Whitaker

et al.

Neuron, Journal Year: 2019, Volume and Issue: 102(1), P. 105 - 119.e8

Published: Feb. 18, 2019

Language: Английский

Citations

85
Characterization of Kcnk3 -Mutated Rat, a Novel Model of Pulmonary Hypertension DOI Open Access
Mélanie Lambert, Véronique Capuano, Angèle Boët

et al.

Circulation Research, Journal Year: 2019, Volume and Issue: 125(7), P. 678 - 695

Published: July 26, 2019

Rationale: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier + channel, have been identified pulmonary patients. Objective: We demonstrated that dysfunction common to heritable and nonheritable experimental (PH). Finally, not functional mouse vasculature. Methods Results: Using CRISPR/Cas9 technology, we generated 94 bp out frame deletion exon 1 Kcnk3 gene characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, molecular levels decipher cellular mechanisms associated with loss KCNK3. patch-clamp technique, validated our transgenic strategy by demonstrating absence current freshly isolated smooth muscle cells from -mutated rats. At 4 months age, echocardiographic parameters revealed shortening artery acceleration time elevation right ventricular systolic pressure. developed more PH than wild-type after monocrotaline exposure or chronic hypoxia exposure. -mutation induced lung distal neomuscularization perivascular extracellular matrix activation. Lungs were overactivation ERK1/2 (extracellular signal–regulated kinase1-/2), AKT (protein kinase B), SRC, overexpression HIF1-α (hypoxia-inducible factor-1 α), survivin, VWF (Von Willebrand factor). Linked plasma membrane depolarization, reduced endothelial-NOS expression desensitization endothelial-derived hyperpolarizing factor, presented predisposition vasoconstriction arteries sildenafil-induced relaxation. Moreover, showed strong alteration cardiomyocyte excitability. age-dependent low serum-albumin concentration. Conclusions: established first rat model PH. Our results confirm key event pathogenesis. This presents new opportunities for understanding initiating testing biologically relevant therapeutic molecules context

Language: Английский

Citations

85
CRISPR in livestock: From editing to printing DOI Open Access
Alejo Menchaca,

P. C. dos Santos-Neto,

Ana Paula Mulet

et al.

Theriogenology, Journal Year: 2020, Volume and Issue: 150, P. 247 - 254

Published: Jan. 29, 2020

Language: Английский

Citations

71
Genetics and pathophysiology of mitral valve prolapse DOI Creative Commons
Constance Delwarde, Romain Capoulade, Jean Mérot

et al.

Frontiers in Cardiovascular Medicine, Journal Year: 2023, Volume and Issue: 10

Published: Feb. 16, 2023

Mitral valve prolapse (MVP) is a common condition affecting 2–3% of the general population, and most complex form pathology, with complication rate up to 10–15% per year in advanced stages. Complications include mitral regurgitation which can lead heart failure atrial fibrillation, but also life-threatening ventricular arrhythmia cardiovascular death. Sudden death has been recently brought forefront MVP disease, increasing complexity management suggesting that not properly understood. occur as part syndromic conditions such Marfan syndrome, non-syndromic, isolated or familial. Although specific X-linked was initially identified, autosomal dominant inheritance appears be primary mode transmission. stratified into myxomatous degeneration (Barlow), fibroelastic deficiency, Filamin A-related MVP. While FED still considered degenerative disease associated aging, FlnA-MVP are recognized familial pathologies. Deciphering genetic defects work progress; although FLNA , DCHS1 DZIP1 have identified causative genes forms thanks approaches, they explain only small proportion In addition, genome-wide association studies revealed important role variants development MVP, agreement high prevalence this population. Furthermore, potential link between type cardiomyopathy considered. Animal models allow advance pathophysiological knowledge particular those easily manipulated express defect humans detailed. Corroborated by data animal models, main pathways briefly addressed. Finally, counseling context

Language: Английский

Citations

25
Nanoparticles-mediated CRISPR/Cas9 delivery: Recent advances in cancer treatment DOI
Shahin Aghamiri,

Sam Talaei,

Afshin Abdi Ghavidel

et al.

Journal of Drug Delivery Science and Technology, Journal Year: 2020, Volume and Issue: 56, P. 101533 - 101533

Published: Jan. 23, 2020

Language: Английский

Citations

51
Regulation of Proteins to the Cytosol Using Delivery Systems with Engineered Polymer Architecture DOI
Jessica A. Kretzmann, David C. Luther, Cameron W. Evans

et al.

Journal of the American Chemical Society, Journal Year: 2021, Volume and Issue: 143(12), P. 4758 - 4765

Published: March 11, 2021

Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction defined quantities into the cell. Most applications require that delivered has access to cytosol, either for activity or as a gateway other organelles such nucleus. The vast majority vehicles employ an endosomal pathway however, efficient release entrapped cargo from endosome remains challenge. Recent research made significant advances toward cytosolic proteins using polymers, but influence polymer architecture on is yet be investigated. Here, we developed family dendronized polymers enable systematic alterations charge density structure. We demonstrate while modulation surface functionality effect overall efficiency, rate can highly regulated by manipulating architecture. Notably, show large, multivalent structures cause slower sustained release, rigid spherical result in rapid burst release.

Language: Английский

Citations

42