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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 -
Neuroscience Letters Feb 2021Stress-induced activation of locus coeruleus (LC)-norepinephrine (NE) projections to the prefrontal cortex are thought to promote cognitive responses to stressors. LC...
Stress-induced activation of locus coeruleus (LC)-norepinephrine (NE) projections to the prefrontal cortex are thought to promote cognitive responses to stressors. LC activation by stressors is modulated by endogenous opioids that restrain LC activation and facilitate a return to baseline activity upon stress termination. Sex differences in this opioid influence could be a basis for sex differences in stress vulnerability. Consistent with this, we recently demonstrated that μ-opioid receptor (MOR) expression is decreased in the female rat LC compared to the male LC, and this was associated with sexually distinct consequences of activating MOR in the LC on cognitive flexibility. Given that the LC-NE system affects cognitive flexibility through its projections to the medial prefrontal cortex (mPFC), the present study quantified and compared the effects of LC-MOR activation on mPFC neural activity in male and female rats. Local field potential (LFPs) were recorded from the mPFC of freely behaving male and female rats before and following local LC microinjection of the MOR agonist, DAMGO, or vehicle. Intra-LC DAMGO altered the LFP power spectrum selectively in male but not female rats, resulting in a time-dependent increase in the power in delta and alpha frequency bands. LC microinfusion of ACSF had no effect on either sex. Together, the results are consistent with previous evidence for decreased MOR function in the female rat LC and demonstrate that this translates to a diminished effect on cortical activity that can account for sex differences in cognitive consequences. Decreased LC-MOR function in females could contribute to greater stress-induced activation of the LC and increased vulnerability of females to hyperarousal symptoms of stress-related neuropsychiatric pathologies.
Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Locus Coeruleus; Male; Microinjections; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Sex Characteristics; Synaptic Transmission
PubMed: 33482313
DOI: 10.1016/j.neulet.2021.135651 -
Neuropharmacology Oct 2018Hemorrhages occurring within the thalamus lead to a pain syndrome. Clinical treatment of thalamic pain is ineffective, at least in part, due to the elusive mechanisms...
Hemorrhages occurring within the thalamus lead to a pain syndrome. Clinical treatment of thalamic pain is ineffective, at least in part, due to the elusive mechanisms that underlie the induction and maintenance of thalamic pain. The present study investigated the possible contribution of a protein-protein interaction between postsynaptic density protein 95 (PSD-95) and neuronal nitric oxide synthase (nNOS) to thalamic pain in mice. Thalamic hemorrhage was induced by microinjection of type IV collagenase into unilateral ventral posterior medial/lateral nuclei of the thalamus. Pain hypersensitivities, including mechanical allodynia, heat hyperalgesia, and cold allodynia, appeared at day 1 post-microinjection, reached a peak 5-7 days post-microinjection, and persisted for at least 28 days post-microinjection on the contralateral side. Systemic pre-treatment (but not post-treatment) of ZL006, a small molecule that disrupts PSD-95-nNOS interaction, alleviated these pain hypersensitivities. This effect is dose-dependent. Mechanistically, ZL006 blocked the hemorrhage-induced increase of binding of PSD-95 with nNOS and membrane translocation of nNOS in thalamic neurons. Our findings suggest that the protein-protein interaction between PSD-95 and nNOS in the thalamus plays a significant role in the induction of thalamic pain. This interaction may be a promising therapeutic target in the clinical management of hemorrhage-induced thalamic pain.
Topics: Aminosalicylic Acids; Animals; Benzylamines; Cerebral Hemorrhage; Collagen Type IV; Disks Large Homolog 4 Protein; Dose-Response Relationship, Drug; Male; Mice; Microinjections; Neuralgia; Nitric Oxide Synthase Type I; Pain Measurement; Protein Binding; Thalamus
PubMed: 30193808
DOI: 10.1016/j.neuropharm.2018.09.003 -
Stroke Jul 2021Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N6-methyladenosine modification is an additional layer of gene regulation....
BACKGROUND AND PURPOSE
Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N6-methyladenosine modification is an additional layer of gene regulation. Whether FTO (fat-mass and obesity-associated protein), an N6-methyladenosine demethylase, participates in hemorrhage-induced thalamic pain is unknown.
METHODS
Expression of Fto mRNA and protein was assessed in mouse thalamus after hemorrhage caused by microinjection of Coll IV (type IV collagenase) into unilateral thalamus. Effect of intraperitoneal administration of meclofenamic acid (a FTO inhibitor) or microinjection of adeno-associated virus 5 (AAV5) expressing Cre into the thalamus of Ftofl/fl mice on the Coll IV microinjection–induced TLR4 (Toll-like receptor 4) upregulation and nociceptive hypersensitivity was examined. Effect of thalamic microinjection of AAV5 expressing Fto (AAV5-Fto) on basal thalamic TLR4 expression and nociceptive thresholds was also analyzed. Additionally, level of N6-methyladenosine in Tlr4 mRNA and its binding to FTO or YTHDF2 (YTH N6-methyladenosine RNA binding protein 2) were observed.
RESULTS
FTO was detected in neuronal nuclei of thalamus. Level of FTO protein, but not mRNA, was time-dependently increased in the ipsilateral thalamus on days 1 to 14 after Coll IV microinjection. Intraperitoneal injection of meclofenamic acid or adeno-associated virus-5 expressing Cre microinjection into Ftofl/fl mouse thalamus attenuated the Coll IV microinjection–induced TLR4 upregulation and tissue damage in the ipsilateral thalamus and development and maintenance of nociceptive hypersensitivities on the contralateral side. Thalamic microinjection of AAV5-Fto increased TLR4 expression and elicited hypersensitivities to mechanical, heat and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding to Tlr4 mRNA, an FTO-dependent loss of N6-methyladenosine sites in Tlr4 mRNA and a reduction in the binding of YTHDF2 to Tlr4 mRNA in the ipsilateral thalamus.
CONCLUSIONS
Our findings suggest that FTO participates in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons. FTO may be a potential target for the treatment of this disorder.
Topics: Adenosine; Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Animals; Cerebral Hemorrhage; Gene Knockdown Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microinjections; Neuralgia; Neurons; Thalamus; Toll-Like Receptor 4
PubMed: 34102854
DOI: 10.1161/STROKEAHA.121.034173 -
Current Protocols in Human Genetics Oct 2016Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models. While efficient, the injected DNA...
Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models. While efficient, the injected DNA integrates randomly into the genome, leading to potential disruption of endogenous genes or regulatory elements, variation in copy number, or integration into heterochromatic regions that inhibit transgene expression. A recently developed method addresses such pitfalls of traditional transgenesis by targeting the transgene to predetermined sites in the genome that can safely harbor exogenous DNA. This method, called Pronuclear Injection-based Targeted Transgenesis (PITT), employs an enzymatic transfer of exogenous DNA from a donor vector to a previously created landing-pad site in the mouse genome. DNA transfer is achieved using molecular tools such as the Cre-LoxP recombinase and PhiC31-attB/P integrase systems. Here, we provide protocols for performing PITT and an overview of the current PITT tools available to the research community. © 2016 by John Wiley & Sons, Inc.
Topics: Animals; Attachment Sites, Microbiological; Cell Nucleus; DNA; Embryo, Mammalian; Female; Gene Targeting; Gene Transfer Techniques; Genome; Integrases; Male; Mice; Microinjections; Recombination, Genetic; Transgenes
PubMed: 27727435
DOI: 10.1002/cphg.23 -
Journal of Visualized Experiments : JoVE Dec 2017The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination,...
The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.
Topics: Animals; Female; Genome; Male; Microinjections; Wasps
PubMed: 29364231
DOI: 10.3791/56990 -
Current Protocols in Cell Biology Dec 2018CRISPR/Cas9 technology has revolutionized genome editing in mice, allowing for simple and rapid development of knockouts and knockins. CRISPR relies on small guide RNAs...
CRISPR/Cas9 technology has revolutionized genome editing in mice, allowing for simple and rapid development of knockouts and knockins. CRISPR relies on small guide RNAs that direct the RNA-guided nuclease Cas9 to a designated genomic site using ∼20 bp of corresponding sequence. Cas9 then creates a double-strand break in the targeted loci that is either patched in an error-prone fashion to produce a frame-shift mutation, a knockout, or is repaired by recombination with donor DNA containing homology arms, a knockin. This protocol covers the techniques needed to rapidly generate knockout and knockin mice with CRISPR via microinjection of Cas9, the guide RNA, and possible donor DNA into the mouse zygote. © 2018 by John Wiley & Sons, Inc.
Topics: Animals; CRISPR-Associated Protein 9; CRISPR-Cas Systems; DNA End-Joining Repair; Electroporation; Embryo, Mammalian; Gene Editing; Genetic Engineering; Genotyping Techniques; INDEL Mutation; Mice; Mice, Knockout; Microinjections; Point Mutation; RNA, Guide, CRISPR-Cas Systems
PubMed: 30178917
DOI: 10.1002/cpcb.57 -
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 -
Biological & Pharmaceutical Bulletin 2017The possibility of using dissolving microneedles (DMs) as a skin allergy test device was studied in rats. Poly-L-arginine was used as a model allergen. Dextran was used...
The possibility of using dissolving microneedles (DMs) as a skin allergy test device was studied in rats. Poly-L-arginine was used as a model allergen. Dextran was used to prepare three kinds of DM array chips containing different doses of poly-L-arginine: 17.1±0.5 µg (low-dose DM), 42.2±0.8 µg (medium-dose DM), and 87.4±1.1 µg (high-dose DM); each 1.0 cm chip contained 300 DMs. The mean lengths of the low-, medium-, and high-dose DM were 489±3, 485±3, and 492±1 µm and mean diameters of the base were 301±2, 299±1, and 299±2 µm, respectively. Furthermore, for the low-, medium-, and high-dose DM, the administered doses of poly-L-arginine were estimated to be 9.3±1.9, 31.1±1.3, and 61.9±4.7 µg and the scratching behavior per 30 min was 9.8±3.4, 60.4±8.3, and 95.7±10.6 times, respectively. These results demonstrate the dose dependence of the immunoreactivity of the poly-L-arginine DMs, suggesting that DMs can be used an alternative skin allergy device.
Topics: Administration, Cutaneous; Allergens; Animals; Dose-Response Relationship, Drug; Hypersensitivity; Male; Microinjections; Needles; Rats; Rats, Wistar; Skin Absorption; Skin Tests
PubMed: 28381808
DOI: 10.1248/bpb.b16-00768 -
Scientific Reports Jul 2015Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of...
Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model.
Topics: Animals; Carbon; Embryo, Nonmammalian; Female; Fluorescent Dyes; Male; Microinjections; Microscopy, Fluorescence; Quantum Dots; Zebrafish
PubMed: 26135470
DOI: 10.1038/srep11835