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Theriogenology Aug 2021The ability to efficiently introduce site-specific genetic modifications to the mammalian genome has been dramatically improved with the use of the CRISPR/Cas9 system....
Effect of ARTEMIS (DCLRE1C) deficiency and microinjection timing on editing efficiency during somatic cell nuclear transfer and in vitro fertilization using the CRISPR/Cas9 system.
The ability to efficiently introduce site-specific genetic modifications to the mammalian genome has been dramatically improved with the use of the CRISPR/Cas9 system. CRISPR/Cas9 is a powerful tool used to generate genetic modifications by causing double-strand breaks (DSBs) in DNA. Artemis (ART; also known as DCLRE1C), is a nuclear protein and is essential for DSB end joining in DNA repair via the canonical non-homologous end joining (c-NHEJ) pathway. In this work, we tested whether ART deficiency affects DNA repair following CRISPR/Cas9 induced DSBs in somatic cells. We also demonstrated the effect of microinjection timing on embryo developmental ability and gene targeting efficiency of CRISPR/Cas9 system to disrupt the interleukin 2 receptor subunit gamma (IL2RG) locus using porcine in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryos. In comparison to non-injected controls, CRISPR/Cas9 injection of IVF derived zygotes at 4 h and 8 h after fertilization did not impact cleavage and blastocyst rate. Gene modification rate was observed to be higher, 53.3% (9/16) in blastocysts injected 4 h post-fertilization as compared to 11.1% (1/9) in blastocysts injected 8 h post-fertilization. Microinjection 8 h after chemical activation of SCNT derived embryos decreased blastocyst development rate compared to non-injected controls but showed a higher gene modification efficiency of 66.7% as compared to 25% in the 4 h post-activation injection group. Furthermore, we observed that male ART and ART porcine fetal fibroblast (pFF) cells showed lower modification rates (2.5% and 1.9%, respectively) as compared to the ART intact cell line (8.3%). Interestingly, the female ART and ART pFF cells had modification rates (4.2% and 10.1%, respectively) similar to those seen in the ART intact cells. This study demonstrates the complex effect of various parameters such as microinjection timing and ART deficiency on gene editing efficiency in in vitro derived porcine embryos.
Topics: Animals; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Female; Fertilization in Vitro; Gene Editing; Male; Microinjections; Swine
PubMed: 34004455
DOI: 10.1016/j.theriogenology.2021.04.003 -
ACS Sensors May 2024Bioanalyte collection by blood draw is a painful process, prone to needle phobia and injuries. Microneedles can be engineered to penetrate the epidermal skin barrier and... (Review)
Review
Bioanalyte collection by blood draw is a painful process, prone to needle phobia and injuries. Microneedles can be engineered to penetrate the epidermal skin barrier and collect analytes from the interstitial fluid, arising as a safe, painless, and effective alternative to hypodermic needles. Although there are plenty of reviews on the various types of microneedles and their use as drug delivery systems, there is a lack of systematization on the application of polymeric microneedles for diagnosis. In this review, we focus on the current state of the art of this field, while providing information on safety, preclinical and clinical trials, and market distribution, to outline what we believe will be the future of health monitoring.
Topics: Needles; Humans; Polymers; Drug Delivery Systems; Animals; Microinjections; Monitoring, Physiologic
PubMed: 38654679
DOI: 10.1021/acssensors.4c00612 -
Methods in Cell Biology 2019To characterize the complex regulatory control of gene expression using fluorescent protein reporters, it is often necessary to analyze large genomic regions. Bacteria... (Review)
Review
To characterize the complex regulatory control of gene expression using fluorescent protein reporters, it is often necessary to analyze large genomic regions. Bacteria artificial chromosome (BAC) vectors, which are able to support DNA fragments of up to 300kb, provide stable platforms for experimental manipulation. Using phage-based systems of homologous recombination, BACs can be efficiently engineered for a variety of aims. These include expressing fluorescent proteins to delineate gene expression boundaries using high-resolution, in vivo microscopy, tracing cell lineages using stable fluorescent proteins, perturbing endogenous protein function by expressing dominant negative forms, interfering with development by mis-expressing transcription factors, and identifying regulatory regions through deletion analysis. Here, we present a series of protocols for identifying BAC clones that contain genes of interest, modifying BACs for use as reporter constructs, and preparing BAC DNA for microinjection into fertilized eggs. Although the protocols here are tailored for use in echinoderm embryonic and larval stages, these methods are easily adaptable for use in other transgenic systems. As fluorescent protein technology continues to expand, so do the potential applications for recombinant BACs.
Topics: Animals; Chromosomes, Artificial, Bacterial; Echinodermata; Gene Expression Regulation, Developmental; Genetic Vectors; Genome; Genomics; Microinjections; Recombination, Genetic; Regulatory Sequences, Nucleic Acid
PubMed: 30948008
DOI: 10.1016/bs.mcb.2019.01.004 -
Methods in Molecular Biology (Clifton,... 2016Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of...
Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.
Topics: Animals; Chronic Pain; Dependovirus; Ganglia, Spinal; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Male; Microinjections; Rats; Rats, Sprague-Dawley; Transduction, Genetic
PubMed: 26611592
DOI: 10.1007/978-1-4939-3271-9_18 -
ASN Neuro 2021Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson's disease (PD). Importantly, neuronal loss...
Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson's disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.
Topics: Adrenergic Neurons; Animals; Dopamine beta-Hydroxylase; Female; Locus Coeruleus; Male; Mice; Mice, Transgenic; Microinjections; Norepinephrine; Parkinsonian Disorders; Tyrosine 3-Monooxygenase; Vesicular Monoamine Transport Proteins
PubMed: 33940943
DOI: 10.1177/17590914211009730 -
Journal of Visualized Experiments : JoVE Feb 2017Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each...
Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.
Topics: Animals; Biological Transport; Cell Communication; Cell Differentiation; Cells, Cultured; Epithelial Cells; Fluorescent Dyes; Gap Junctions; Isoquinolines; Mice; Microinjections; Microscopy, Fluorescence; Models, Animal
PubMed: 28287521
DOI: 10.3791/50836 -
Current Neuropharmacology 2015Rats emit high-frequency 50-kHz ultrasonic vocalizations (USV) in appetitive situations like social interactions. Drugs of abuse are probably the most potent non-social... (Review)
Review
Rats emit high-frequency 50-kHz ultrasonic vocalizations (USV) in appetitive situations like social interactions. Drugs of abuse are probably the most potent non-social elicitors of 50-kHz USV, possibly reflecting their euphorigenic properties. Psychostimulants induce the strongest elevation in 50-kHz USV emission, particularly amphetamine (AMPH), either when applied systemically or locally into the nucleus accumbens (Nacc). Emission of AMPH-induced 50-kHz USV depends on test context, such as the presence of conspecifics, and can be manipulated pharmacologically by targeting major neurotransmitter systems, including dopamine (DA), noradrenaline (NA), and serotonin (5-HT), but also protein kinase C (PKC) signaling. Several D1 and D2 receptor antagonists, as well as typical and atypical antipsychotics block the AMPH-induced elevation in 50-kHz USV. Inhibiting D1 and D2 receptors in the Nacc abolishes AMPH-induced 50-kHz USV, indicating a key role for this brain area. NA neurotransmission also regulates AMPH-induced 50-kHz USV emission given that α 1 receptor antagonists and α 2 receptor agonists exert attenuating effects. Supporting the involvement of the 5-HT system, AMPH-induced 50-kHz USV are attenuated by 5-HT2C receptor activation, whereas 5-HT2C receptor antagonism leads to the opposite effect. Finally, treatment with lithium, tamoxifen, and myricitrin was all found to result in a complete abolishment of the AMPH-induced increase in 50-kHz USV, suggesting the involvement of PKC signaling. Neurotransmitter systems involved in AMPH-induced 50-kHz USV emission only partially overlap with other AMPH-induced behaviors like hyperlocomotion. The validity of AMPHinduced 50-kHz USV as a preclinical model for neuropsychiatric disorders is discussed, particularly with relevance to altered drive and mood seen in bipolar disorder.
Topics: Amphetamine; Animals; Antimanic Agents; Central Nervous System Stimulants; Dose-Response Relationship, Drug; Environment; Microinjections; Neurotransmitter Agents; Rats; Ultrasonics; Vocalization, Animal
PubMed: 26411764
DOI: 10.2174/1570159x1302150525124408 -
Journal of Visualized Experiments : JoVE Aug 2014This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection...
This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis. The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV). For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red fluorescence, illustrating transfection success. Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.
Topics: Animals; Electroporation; Genes, Reporter; Green Fluorescent Proteins; Male; Mice; Microinjections; Plasmids; Testis; Transfection
PubMed: 25177859
DOI: 10.3791/51802 -
Current Protocols in Nucleic Acid... Mar 2017Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein...
Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein-mediated catalysis-nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos. © 2017 by John Wiley & Sons, Inc.
Topics: Cytosol; Electroporation; Endocytosis; Gene Expression Regulation; Gene Knockdown Techniques; Microinjections; Morpholinos; Peptides; RNA
PubMed: 28252184
DOI: 10.1002/cpnc.21 -
Cellular and Molecular Gastroenterology... 2018The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to...
BACKGROUND & AIMS
The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host-microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling.
METHODS
A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe.
RESULTS
CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein- or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity.
CONCLUSIONS
High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.
Topics: Animals; Bifidobacterium adolescentis; Colon; Escherichia coli; Feces; Gastrointestinal Microbiome; Gastrointestinal Tract; Humans; Male; Mice; Microinjections; Organoids; Single-Cell Analysis; Video Recording; Yersinia pseudotuberculosis
PubMed: 30123820
DOI: 10.1016/j.jcmgh.2018.05.004