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Theriogenology Jul 2022Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This...
Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs.
Topics: Animals; CRISPR-Cas Systems; Calpain; Electroporation; Gene Editing; Insemination; Microinjections; Oocytes; Swine
PubMed: 35500431
DOI: 10.1016/j.theriogenology.2022.04.012 -
Toxins Mar 2022Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are and Sterigmatocystin (STC), another mycotoxin, shares its...
Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are and Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment.
Topics: Aflatoxin B1; Aflatoxins; Animals; Escherichia coli; Microinjections; Sterigmatocystin; Zebrafish
PubMed: 35448861
DOI: 10.3390/toxins14040252 -
Neuropsychopharmacology : Official... Jun 2018Despite the cost to both individual and society, alcohol use disorders (AUDs) remain a major health risk within society, and both relapse and heavy drinking are still...
Despite the cost to both individual and society, alcohol use disorders (AUDs) remain a major health risk within society, and both relapse and heavy drinking are still poorly controlled with current medications. Here we demonstrate for the first time that a centrally active and selective negative allosteric modulator for the rat M muscarinic acetylcholine receptor (mAChR), ML375, decreases ethanol self-administration and attenuates cue-induced reinstatement of ethanol seeking in ethanol-preferring (iP) rats. Importantly, ML375 did not affect sucrose self-administration or general locomotor activity indicative of a selective effect on ethanol seeking. Based on the expression profile of M mAChRs in the brain and the distinct roles different aspects of the dorsal striatum have on long-term and short-term ethanol use, we studied whether intra-striatal microinjection of ML375 modulated ethanol intake in rats. We show in iP rats with an extensive history of ethanol intake that intra-dorsolateral (DL), but not intra-dorsomedial, striatal injections of ML375 reduced ethanol self-administration to a similar extent as the nicotinic acetylcholine receptor ligand varenicline, which has preclinical and clinical efficacy in reducing the reinforcing effects of ethanol. These data implicate the DL striatum as a locus for the effects of cholinergic-acting drugs on ethanol seeking in rats with a history of long-term ethanol use. Accordingly, we demonstrate in rats that selectively targeting the M mAChR can modulate both voluntary ethanol intake and cue-induced ethanol seeking and thereby provide direct evidence that the M mAChR is a potential novel target for pharmacotherapies aimed at treating AUDs.
Topics: Animals; Conditioning, Operant; Corpus Striatum; Cues; Drug-Seeking Behavior; Ethanol; Imidazoles; Indoles; Locomotion; Male; Microinjections; Rats; Receptor, Muscarinic M5; Self Administration; Sucrose; Varenicline
PubMed: 29483658
DOI: 10.1038/s41386-017-0007-3 -
Brain Research Apr 2022The central nucleus of the amygdala (CNA) projects to brainstem regions that generate and regulate rapid eye movement sleep (REM). We used optogenetics to assess the...
The central nucleus of the amygdala (CNA) projects to brainstem regions that generate and regulate rapid eye movement sleep (REM). We used optogenetics to assess the influence of CNA inputs into reticularis pontis oralis (RPO), pedunculopontine tegmentum (PPT) and nucleus subcoeruleus (SubC) on dark period sleep. We compared these results to effects of microinjections into CNA of the GABA agonist, muscimol (MUS, inhibition of cell bodies) and tetrodotoxin (TTX, inhibition of cell bodies and fibers of passage). For optogenetics, male Wistar rats received excitatory (AAV5-EF1a-DIO -hChR2(H134R)-EYFP) or inhibitory (AAV-EF1a-DIO-eNpHR3.0-EYFP; DIO-eNpHR3.0) opsins into CNA and AAV5-EF1a-mCherry-IRES-WGA-Cre into RPO, PPT, or SubC. This enabled only CNA neurons synaptically connected to each region to express opsin. Optic cannulae for light delivery into CNA and electrodes for determining sleep were implanted. Sleep was recorded with and without blue or amber light stimulation of CNA. Separate rats received MUS or TTX into CNA prior to recording sleep. Optogenetic activation of CNA neurons projecting to RPO enhanced REM and did not alter non-REM (NREM) whereas activation of CNA neurons projecting to PPT or SubC did not significantly affect sleep. Inhibition of CNA neurons projecting to any region did not significantly alter sleep. TTX inactivation of CNA decreased REM and increased NREM whereas muscimol inactivation did not significantly alter sleep. Thus, the amygdala can regulate decreases and increases in REM, and RPO is important for CNA promotion of REM. Fibers passing through CNA, likely from the basolateral nucleus of the amygdala, also play a role in regulating sleep.
Topics: Animals; Central Amygdaloid Nucleus; Electroencephalography; Male; Microinjections; Muscimol; Optogenetics; Rats; Rats, Wistar; Sleep; Tetrodotoxin; Wakefulness
PubMed: 35131286
DOI: 10.1016/j.brainres.2022.147816 -
Cold Spring Harbor Protocols Apr 2018The oocyte is a specialized single cell of colossal size (>1 mm diameter) that is highly amenable for microinjection and a stalwart model for heterologous expression....
The oocyte is a specialized single cell of colossal size (>1 mm diameter) that is highly amenable for microinjection and a stalwart model for heterologous expression. Oocytes are easily obtainable, robust in vitro, and faithfully express injected constructs. Their large size translational capacity provides a huge canvas for observing and recording integrated cellular responses-from studies of single molecules within single cells to medium-throughput drug-screening applications. Most eukaryotic promoters suffice for expression, and the oocyte can functionally express proteins from many diverse organisms. This protocol provides a basic introduction for scientists keen to perform nuclear microinjections of cDNA constructs. These are easy methods to master, do not require elaborate equipment, and make accessible a wonderful model cell system for studying signaling, transport, cell architecture, and protein function.
Topics: Animals; DNA, Complementary; Female; Gene Expression; Microinjections; Molecular Biology; Oocytes; Xenopus laevis
PubMed: 29208643
DOI: 10.1101/pdb.prot096990 -
Scientific Reports Mar 2019A comprehensive experimental system for Japanese anchovy, a promising candidate model organism for marine teleosts, was established. Through the design of a...
A comprehensive experimental system for Japanese anchovy, a promising candidate model organism for marine teleosts, was established. Through the design of a rearing/spawning facility that controls the photoperiod and water temperature, one-cell eggs were continuously obtained shortly after spawning throughout the rearing period. The stages of eggs are indispensable for microinjection experiments, and we developed an efficient and robust microinjection system for the Japanese anchovy. Embryos injected with GFP mRNA showed strong whole-body GFP fluorescence and the survival rates of injected- and non-injected embryos were not significantly different, 87.5% (28 in 32 embryos) and 90.0% (45 in 50 embryos), respectively. We verified that the Tol2 transposon system, which mediates gene transfer in vertebrates, worked efficiently in the Japanese anchovy using the transient transgenesis protocol, with GFP or DsRed as the reporter gene. Finally, we confirmed that genome-editing technologies, namely Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9, were applicable to the Japanese anchovy. In practice, specific gene-disrupted fishes were generated in the F generation. These results demonstrated the establishment of a basic, yet comprehensive, experimental system, which could be employed to undertake experiments using the Japanese anchovy as a model organism for marine teleost fish.
Topics: Animals; Animals, Genetically Modified; CRISPR-Cas Systems; DNA Transposable Elements; Embryo, Nonmammalian; Fishes; Gene Editing; Microinjections; Models, Animal; Seawater; Transcription Activator-Like Effector Nucleases
PubMed: 30894668
DOI: 10.1038/s41598-019-41468-8 -
Advanced Science (Weinheim,... Aug 2021A closed-loop system that can mini-invasively track blood glucose and intelligently treat diabetes is in great demand for modern medicine, yet it remains challenging to...
A closed-loop system that can mini-invasively track blood glucose and intelligently treat diabetes is in great demand for modern medicine, yet it remains challenging to realize. Microneedles technologies have recently emerged as powerful tools for transdermal applications with inherent painlessness and biosafety. In this work, for the first time to the authors' knowledge, a fully integrated wearable closed-loop system (IWCS) based on mini-invasive microneedle platform is developed for in situ diabetic sensing and treatment. The IWCS consists of three connected modules: 1) a mesoporous microneedle-reverse iontophoretic glucose sensor; 2) a flexible printed circuit board as integrated and control; and 3) a microneedle-iontophoretic insulin delivery component. As the key component, mesoporous microneedles enable the painless penetration of stratum corneum, implementing subcutaneous substance exchange. The coupling with iontophoresis significantly enhances glucose extraction and insulin delivery and enables electrical control. This IWCS is demonstrated to accurately monitor glucose fluctuations, and responsively deliver insulin to regulate hyperglycemia in diabetic rat model. The painless microneedles and wearable design endows this IWCS as a highly promising platform to improve the therapies of diabetic patients.
Topics: Animals; Diabetes Mellitus, Experimental; Disease Models, Animal; Hypoglycemic Agents; Insulin; Iontophoresis; Male; Microinjections; Needles; Rats; Rats, Sprague-Dawley
PubMed: 34081407
DOI: 10.1002/advs.202100827 -
PLoS Neglected Tropical Diseases Sep 2018Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances...
BACKGROUND
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis.
METHODOLOGY/PRINCIPAL FINDINGS
Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed.
CONCLUSION AND SIGNIFICANCE
The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.
Topics: Animals; CRISPR-Associated Protein 9; Clustered Regularly Interspaced Short Palindromic Repeats; Embryo, Nonmammalian; Entomology; Female; Gene Editing; Insect Vectors; Male; Microinjections; Psychodidae
PubMed: 30180160
DOI: 10.1371/journal.pntd.0006769 -
Veterinary Medicine and Science Jul 2021Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental...
Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental genes has a critical role in growth and development of embryo, we determined the expression pattern of pluripotency, trophectoderm and imprinting genes in the Rag1 (recombination-activating gene 1) knocked-out blastocysts resulting from microinjection of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) constructs into the zygote cytoplasm of C57bl6 mice. Following microinjection, the embryos were cultured and the gene expression of developed blastocysts and natural blastocysts (Sham and control groups) were evaluated using real-time PCR. The agarose gel to confirm the deletion in the Rag1 gene in Rag1 knocked-out blastocyst. Our results showed that the expression of trophectoderm genes (-TEAD-4 and Cdx2), pluripotency genes (Nanog and Oct-4) and imprinting gene (H19) in the Rag1 knocked-out group was significantly lower compared with the embryos obtained from Natural fertilization. According to these findings, manipulation, embryo culture and microinjection of CRISPR constructs into the zygote cytoplasm of mice led to reduced expression of imprinting, pluripotency and trophectoderm genes. Therefore, the Rag1 knocked-out embryos produced by the CRISPR/Cas9 system are of low quality, which reduces the chances of live birth in these animals and may cause various abnormalities in fetuses.
Topics: Animals; Clustered Regularly Interspaced Short Palindromic Repeats; Embryo, Mammalian; Gene Expression Regulation, Developmental; Genes, Developmental; Homeodomain Proteins; Mice; Mice, Knockout; Microinjections
PubMed: 33955691
DOI: 10.1002/vms3.380 -
BMC Research Notes Jan 2021Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create... (Comparative Study)
Comparative Study
OBJECTIVE
Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos.
RESULTS
First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.
Topics: Animals; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Electroporation; Microinjections; Swine
PubMed: 33407863
DOI: 10.1186/s13104-020-05412-8