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Systems Biology in Reproductive Medicine Jun 2010Echinoderms are closely related to chordates and comprise a major group of invertebrate deuterostomes. They are broadcast spawners and as such, each female accumulates... (Review)
Review
Echinoderms are closely related to chordates and comprise a major group of invertebrate deuterostomes. They are broadcast spawners and as such, each female accumulates millions of eggs and oocytes. These cells are readily isolated, and are often large, clear, and surrounded by accessory cells and extracellular coverings that do not prevent access to the oocyte. Sea star oocytes are stored in prophase of meiosis, and since the natural meiotic stimulus has been identified as 1-methyladenine, these cells can be induced to complete meiotic maturation as individuals, or synchronously en masse. Microinjection and culture of these cells is feasible using quantitative or repetitive methods so that hundreds of oocytes and eggs can be modified each hour. Experimentation on this organism is extensive over a rich history of reproductive and developmental biology so that new investigators can easily incorporate this organism into their repertoire of research. This review will highlight the fundamental protocols to enable a new investigator to perform an array of approaches on this organism, including oocyte isolation, microinjection, and even single cell quantitative PCR.
Topics: Animals; Cell Separation; Environmental Exposure; Female; Male; Microinjections; Models, Animal; Oocytes; Reproduction; Spermatozoa; Starfish
PubMed: 20536323
DOI: 10.3109/19396361003674879 -
STAR Protocols Dec 2020Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the...
Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Genetic Engineering; Genome; Genomics; Microinjections; Mutagenesis, Site-Directed; RNA, Guide, CRISPR-Cas Systems; Sequence Deletion; Zebrafish
PubMed: 33377102
DOI: 10.1016/j.xpro.2020.100208 -
Expert Review of Vaccines May 2012In recent years with the threat of pandemic influenza and other public health needs, alternative vaccination methods other than intramuscular immunization have received... (Review)
Review
In recent years with the threat of pandemic influenza and other public health needs, alternative vaccination methods other than intramuscular immunization have received great attention. The skin and mucosal surfaces are attractive sites probably because of both noninvasive access to the vaccine delivery and unique immunological responses. Intradermal vaccines using a microinjection system (BD Soluvia(TM)) and intranasal vaccines (FluMist®) are licensed. As a new vaccination method, solid microneedles have been developed using a simple device that may be suitable for self-administration. Because coated microneedle influenza vaccines are administered in the solid state, developing formulations maintaining the stability of influenza vaccines is an important issue to be considered. Marketable microneedle devices and clinical trials remain to be developed. Other alternative mucosal routes such as oral and intranasal delivery systems are also attractive for inducing cross-protective mucosal immunity, but effective non-live mucosal vaccines remain to be developed.
Topics: Administration, Intranasal; Adolescent; Adult; Animals; Humans; Immunity, Mucosal; Influenza Vaccines; Injections, Intradermal; Mice; Microinjections; Middle Aged; Needles; Vaccination; Young Adult
PubMed: 22697052
DOI: 10.1586/erv.12.25 -
Advanced Drug Delivery Reviews Mar 2018Conventional systematically-administered drugs distribute evenly throughout the body, get degraded and excreted rapidly while crossing many biological barriers, leaving... (Review)
Review
Conventional systematically-administered drugs distribute evenly throughout the body, get degraded and excreted rapidly while crossing many biological barriers, leaving minimum amounts of the drugs at pathological sites. Controlled drug delivery aims to deliver drugs to the target sites at desired rates and time, thus enhancing the drug efficacy, pharmacokinetics, and bioavailability while maintaining minimal side effects. Due to a number of unique advantages of the recent microfluidic lab-on-a-chip technology, microfluidic lab-on-a-chip has provided unprecedented opportunities for controlled drug delivery. Drugs can be efficiently delivered to the target sites at desired rates in a well-controlled manner by microfluidic platforms via integration, implantation, localization, automation, and precise control of various microdevice parameters. These features accordingly make reproducible, on-demand, and tunable drug delivery become feasible. On-demand self-tuning dynamic drug delivery systems have shown great potential for personalized drug delivery. This review presents an overview of recent advances in controlled drug delivery using microfluidic platforms. The review first briefly introduces microfabrication techniques of microfluidic platforms, followed by detailed descriptions of numerous microfluidic drug delivery systems that have significantly advanced the field of controlled drug delivery. Those microfluidic systems can be separated into four major categories, namely drug carrier-free micro-reservoir-based drug delivery systems, highly integrated carrier-free microfluidic lab-on-a-chip systems, drug carrier-integrated microfluidic systems, and microneedles. Microneedles can be further categorized into five different types, i.e. solid, porous, hollow, coated, and biodegradable microneedles, for controlled transdermal drug delivery. At the end, we discuss current limitations and future prospects of microfluidic platforms for controlled drug delivery.
Topics: Drug Delivery Systems; Humans; Microfluidic Analytical Techniques; Microinjections; Needles
PubMed: 28919029
DOI: 10.1016/j.addr.2017.09.013 -
The American Journal of Managed Care Nov 2022Standard ocular drug delivery methods generally are safe and effective for treating diseases of the eye. However, many routes of administration carry the risk of adverse... (Review)
Review
Standard ocular drug delivery methods generally are safe and effective for treating diseases of the eye. However, many routes of administration carry the risk of adverse effects due to drug exposure to anterior ocular tissues. Additionally, these delivery methods may not result in high and consistent levels of a therapeutic agent delivered to target tissues for diseases affecting the posterior segment of the eye. Injection into the suprachoroidal space (SCS) represents an alternative method of ocular drug delivery to the posterior segment. SCS injection facilitates targeted distribution to affected chorioretinal tissues for potential efficacy benefits, compartmentalization away from unaffected anterior segment tissues for potential safety benefits, and a high degree of bioavailability. Furthermore, the SCS may serve as a drug depot for long-acting drug delivery of small-molecule suspensions. Until recently, drug delivery to the SCS could be achieved only in the operating room setting with anesthetic immobilization of the eye and surgical dissection through the sclera. A novel microneedle device, the SCS Microinjector® (Clearside Biomedical, Inc) was developed to permit physicians to administer therapies safely and reliably into the SCS in the office setting. Successful use of SCS injection has been demonstrated with triamcinolone acetonide injectable suspension (Xipere®, Bausch + Lomb), a novel formulation optimized for use with the SCS Microinjector®. FDA approval of this combination drug and device for the treatment of macular edema associated with uveitis (UME) was based on outcomes from the phase 3 PEACHTREE study (NCT02595398); other important studies included its long-term observational extension (MAGNOLIA; NCT02952001) and an open-label safety study (AZALEA; NCT03097315). The SCS Microinjector® together with triamcinolone acetonide injectable suspension for use in the SCS presents an opportunity for safe and effective drug delivery for the treatment of UME and, potentially, for broader use with alternate medications to treat other ocular diseases that impact chorioretinal tissues (eg, age-related macular degeneration, diabetic retinopathy, choroidal melanoma).
Topics: Humans; Microinjections; Choroid; Triamcinolone Acetonide; Needles; Choroidal Effusions; Observational Studies as Topic
PubMed: 36395492
DOI: 10.37765/ajmc.2022.89270 -
Journal of Plastic, Reconstructive &... Aug 2022Mesenchymal stem cell (MSC)-supplemented acellular nerve allografts (ANA) are a potential strategy to improve the treatment of segmental nerve defects. Prior to clinical...
BACKGROUND
Mesenchymal stem cell (MSC)-supplemented acellular nerve allografts (ANA) are a potential strategy to improve the treatment of segmental nerve defects. Prior to clinical translation, optimal cell delivery methods must be defined. While two techniques, dynamic seeding and microinjection, have been described, the seeding efficiency, cell viability, and distribution of MSCs in ANAs are yet to be compared.
METHODS
Sciatic nerve segments of Sprague-Dawley rats were decellularized, and MSCs were harvested from the adipose tissue of Lewis rats. Cell viability was evaluated after injection of MSCs through a 27-gauge needle at different flow rates (10, 5, and 1 µL/min). MSCs were dynamically seeded or longitudinally injected into ANAs. Cell viability, seeding efficiency, and distribution were evaluated using LIVE/DEAD and MTS assays, scanning electron microscopy, and Hoechst staining.
RESULTS
No statistically significant difference in cell viability after injection at different flow rates was seen. After cell delivery, 84.1 ± 3.7% and 87.8 ± 2.8% of MSCs remained viable in the dynamic seeding and microinjection group, respectively (p = 0.41). The seeding efficiency of microinjection (100.4%±5.6) was significantly higher than dynamic seeding (48.1%±8.6) on day 1 (p = 0.001). Dynamic seeding demonstrated a significantly more uniform cell distribution over the course of the ANA compared to microinjection (p = 0.02).
CONCLUSION
MSCs remain viable after both dynamic seeding and microinjection in ANAs. Higher seeding efficiency was observed with microinjection, but dynamic seeding resulted in a more uniform distribution. In vivo studies are required to assess the effect on gene expression profiles and functional motor outcomes.
Topics: Allografts; Animals; Mesenchymal Stem Cells; Microinjections; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley
PubMed: 35570113
DOI: 10.1016/j.bjps.2022.04.017 -
Developmental Biology Oct 2023Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These...
Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans. While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.
Topics: Animals; Caenorhabditis elegans; Microinjections; Animals, Genetically Modified; Germ Cells; DNA; CRISPR-Cas Systems
PubMed: 37433390
DOI: 10.1016/j.ydbio.2023.07.003 -
Journal of Visualized Experiments : JoVE May 2009An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In...
An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.
Topics: Animals; Green Fluorescent Proteins; Microinjections; Microscopy, Fluorescence; Oligonucleotides, Antisense; RNA, Messenger; Zebrafish
PubMed: 19488022
DOI: 10.3791/1113 -
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 -
STAR Protocols Sep 2021In targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of germline editing is attributed to the syncytial nature...
In targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in , to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).
Topics: Animals; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Gene Editing; Genetic Engineering; Microinjections
PubMed: 34505086
DOI: 10.1016/j.xpro.2021.100748