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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 -
Methods in Cell Biology 2004
Review
Topics: Animals; Echinodermata; Embryo, Nonmammalian; Female; Microelectrodes; Microinjections; Microscopy; Oocytes; Ovum; Syringes
PubMed: 15575609
DOI: 10.1016/s0091-679x(04)74010-8 -
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 -
Journal of Visualized Experiments : JoVE Oct 2016Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of...
Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.
Topics: Animals; Ciona intestinalis; Electroporation; Gene Transfer Techniques; Humans; Microinjections
PubMed: 27805579
DOI: 10.3791/54313 -
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 -
Human Vaccines & Immunotherapeutics Jan 2012Among the several strategies explored for (1) the enhancement of the immune response to influenza immunization, (2) the improvement of the vaccine acceptability and (3)... (Review)
Review
Among the several strategies explored for (1) the enhancement of the immune response to influenza immunization, (2) the improvement of the vaccine acceptability and (3) the overcoming of the egg-dependency for vaccine production, intradermal administration of influenza vaccine emerges as a promising alternative to conventional intramuscular route, thanks to the recent availability of new delivery devices and the perception of advantages in terms of immunogenicity, safety, reduction of antigen content and acceptability. Data from clinical trials performed in children, adults < 60 y and elderly people and post-marketing surveillance demonstrate that actually, licensed intradermal influenza vaccines, Intanza™ 9 and 15 µg and Fluzone™ Intradermal, administered by the microinjection system Soluvia™, show an excellent acceptability, tolerability and safety profile. Formulations containing 9 and 15 μg per strain demonstrate, respectively, comparable and superior immunogenicity than conventional intramuscular vaccines. Licensed intradermal influenza vaccines can be considered a valid alternative to standard intramuscular vaccination offering significant advantages in low-responder populations and helping to increase influenza vaccination coverage rates especially in people with fear of needles or high apprehension associated with annual vaccination.
Topics: Adult; Child, Preschool; Clinical Trials as Topic; Humans; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza Vaccines; Influenza, Human; Injections, Intradermal; Licensure; Microinjections; Middle Aged; Patient Acceptance of Health Care; Treatment Outcome; Vaccination; Young Adult
PubMed: 22293531
DOI: 10.4161/hv.8.1.18419 -
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 -
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 -
Journal of Visualized Experiments : JoVE Dec 2017Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental...
Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.
Topics: Animals; Larva; Microinjections; Zebrafish
PubMed: 29286475
DOI: 10.3791/56498 -
Scientific Reports Jul 2020Gene knockdown techniques are widely used to examine the function of specific genes or proteins. While a variety of techniques are available, a technique commonly used...
Gene knockdown techniques are widely used to examine the function of specific genes or proteins. While a variety of techniques are available, a technique commonly used on mammalian oocytes is mRNA knockdown by microinjection of small interfering RNA (siRNA), with non-specific siRNA injection used as a technical control. Here, we investigate whether and how the microinjection procedure itself affects the transcriptome of bovine oocytes. Injection of non-specific siRNA resulted in differential expression of 119 transcripts, of which 76 were down-regulated. Gene ontology analysis revealed that the differentially regulated genes were enriched in the biological processes of ATP synthesis, molecular transport and regulation of protein polyubiquitination. This study establishes a background effect of the microinjection procedure that should be borne in mind by those using microinjection to manipulate gene expression in oocytes.
Topics: Animals; Cattle; Female; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Microinjections; Oocytes; RNA, Messenger; RNA, Small Interfering; RNA-Seq; Single-Cell Analysis; Transcriptome
PubMed: 32641751
DOI: 10.1038/s41598-020-67603-4