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Methods in Molecular Biology (Clifton,... 2019Microinjection/micromanipulation is more than 100 years old. It is a technique that is instrumental in biomedical research and healthcare. Its longevity lies in its...
Microinjection/micromanipulation is more than 100 years old. It is a technique that is instrumental in biomedical research and healthcare. Its longevity lies in its preciseness in mechanical retrieval, or delivery of biological materials, which in some cases is simply necessary or more effective than other retrieval/delivery means. Microinjection is favored for its straightforwardness in transferring contents from micromolecules to macromolecules and from organelles to cells. Microinjection/micromanipulation has been practiced over the century like an art form. Variations in handlings and instruments can be tolerated to a surprising degree with satisfactory outcomes. Throughout the century, microinjection developed as an indispensable tool along with the evolution of biomedical fields: from transgenics to gene targeting, from animal cloning to human infertility treatment, from nuclease-guided genetic engineering to RNA-guided genome editing (Fig. 1). The birth of the CRISPRology rejuvenated microinjection. For microinjection/micromanipulation, the second century has already begun with the early arrival of computerized instrumentation and lately of the high-throughput nanomanipulators potentially operable by artificial intelligence. As we yin-yang both systemic and precision approaches in research and medicine, microinjection will no doubt continue to find its unique place in the future.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Gene Targeting; Genetic Engineering; History, 20th Century; History, 21st Century; Humans; Microinjections; Micromanipulation; Nanotechnology
PubMed: 30353505
DOI: 10.1007/978-1-4939-8831-0_1 -
Advanced Drug Delivery Reviews Oct 2023In the field of ocular drug delivery, topical delivery remains the most common treatment option for managing anterior segment diseases, whileintraocular injectionsare... (Review)
Review
In the field of ocular drug delivery, topical delivery remains the most common treatment option for managing anterior segment diseases, whileintraocular injectionsare the current gold standard treatment option for treating posterior segment diseases. Nonetheless, topical eye drops are associated with low bioavailability (<5%), and theintravitreal administration procedure is highly invasive, yielding poor patient acceptability. In both cases, frequent administration is currently required. As a result, there is a clear unmet need for sustained drug delivery to the eye, particularly in a manner that can be localised. Microneedles, which are patches containing an array of micron-scale needles (<1 mm), have the potential to meet this need. These platforms can enable localised drug delivery to the eye while enhancing penetration of drug molecules through key ocular barriers, thereby improving overall therapeutic outcomes. Moreover, the minimally invasive manner in which microneedles are applied could provide significant advantages over traditional intravitreal injections regarding patient acceptability. Considering the benefitsofthis novel ocular delivery system, this review provides an in-depth overviewofthe microneedle systems for ocular drug delivery, including the types of microneedles used and therapeutics delivered. Notably, we outline and discuss the current challenges associated with the clinical translation of these platforms and offer opinions on factors which should be considered to improve such transition from lab to clinic.
Topics: Humans; Drug Delivery Systems; Eye; Pharmaceutical Preparations; Needles; Microinjections; Administration, Cutaneous
PubMed: 37678648
DOI: 10.1016/j.addr.2023.115082 -
Small (Weinheim An Der Bergstrasse,... Jun 2024Microneedles (MNs) have maintained their popularity in therapeutic and diagnostic medical applications throughout the past decade. MNs are originally designed to gently... (Review)
Review
Microneedles (MNs) have maintained their popularity in therapeutic and diagnostic medical applications throughout the past decade. MNs are originally designed to gently puncture the stratum corneum layer of the skin and have lately evolved into intelligent devices with functions including bodily fluid extraction, biosensing, and drug administration. MNs offer limited invasiveness, ease of application, and minimal discomfort. Initially manufactured solely from metals, MNs are now available in polymer-based varieties. MNs can be used to create systems that deliver drugs and chemicals uniformly, collect bodily fluids, and are stimulus-sensitive. Although these advancements are favorable in terms of biocompatibility and production costs, they are insufficient for the therapeutic use of MNs. This is the first comprehensive review that discusses individual MN functions toward the evolution and development of smart and multifunctional MNs for a variety of novel and impactful future applications. The study examines fabrication techniques, application purposes, and experimental details of MN constructs that perform multiple functions concurrently, including sensing, drug-molecule release, sampling, and remote communication capabilities. It is highly likely that in the near future, MN-based smart devices will be a useful and important component of standard medical practice for different applications.
Topics: Needles; Humans; Drug Delivery Systems; Animals; Theranostic Nanomedicine; Microinjections
PubMed: 38385813
DOI: 10.1002/smll.202308479 -
Journal of Visualized Experiments : JoVE Sep 2023Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the... (Review)
Review
Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.
Topics: Chick Embryo; Animals; Humans; Chickens; Microinjections; Lens, Crystalline; Lenses; Retroviridae
PubMed: 37677003
DOI: 10.3791/65727 -
Methods in Molecular Biology (Clifton,... 2019Microinjection is a powerful tool for studying embryonic development and analyzing gene functions in fish. This technique was first applied to model species of fish such...
Microinjection is a powerful tool for studying embryonic development and analyzing gene functions in fish. This technique was first applied to model species of fish such as zebrafish and medaka whose egg chorions could be removed or softened before microinjection. Recent progress in genome editing using TALEN and CRISPR has opened the opportunity to analyze gene functions in a much wider range of fish including those important to marine aquaculture. Therefore, application of the microinjection technique is also required in these species. However, the characteristics of fish eggs vary widely between species and several technical difficulties need to be overcome in order to use microinjection in a wider range of species. To obtain consistent results with microinjection, an optimal method has to be developed for each target species. In this chapter, we describe the physical characteristics of the eggs of fish species that have been used in microinjection experiments in our laboratory and detail the microinjection system we developed for fish eggs with a hard chorion, such as those of marine species.
Topics: Animals; Animals, Genetically Modified; Embryonic Development; Female; Fishes; Gene Editing; Microinjections; Ovum
PubMed: 30353531
DOI: 10.1007/978-1-4939-8831-0_27 -
Methods in Cell Biology 2019We describe methods and techniques for introduction of molecular probes in the form of synthetic mRNA by rapid repetitive microinjection into oocytes or early embryos of... (Review)
Review
We describe methods and techniques for introduction of molecular probes in the form of synthetic mRNA by rapid repetitive microinjection into oocytes or early embryos of echinoderms and various invertebrates. Construct assembly is followed by standard kit-based in vitro mRNA synthesis, with slight modifications to optimize expression and clean-up. Variations of a basic microinjection procedures are detailed for echinoderms: starfish oocytes (Patiria miniata or other species), purple urchin (Strongylocentrotus purpuratus) and sand dollar (Dendraster excentricus) zygotes, with notes included for other invertebrate eggs and embryos as well.
Topics: Animals; Embryo, Nonmammalian; Microinjections; Molecular Probes; Oocytes; RNA, Messenger; Sea Urchins; Starfish
PubMed: 30777176
DOI: 10.1016/bs.mcb.2018.10.012 -
Methods in Molecular Biology (Clifton,... 2022The protocol outlined in this chapter describes a detailed procedure for protoplast isolation and transformation using polyethylene glycol (PEG)-mediated transfection...
The protocol outlined in this chapter describes a detailed procedure for protoplast isolation and transformation using polyethylene glycol (PEG)-mediated transfection and DNA microinjection, highlighting also the critical steps associated with the method. Briefly, we will describe the efficient isolation of protoplasts from 3-month-old suspension calli collected at 14 days after cultured. Digestion of the calli with an optimal composition of enzyme solution yielded over 2 × 10 protoplasts/mL with the viability of more than 80%. The concentrations of DNA, PEG, and magnesium chloride and application of heat shock treatment are the crucial determinants for efficient PEG-mediated transfection. Using the optimal PEG transfection conditions, a transfection efficiency of more than 20% could be obtained. At the same time, protoplasts embedded in alginate layer cultured for 3 days and injected with 100 ng/μL of total DNA solution are the optimal factors for microinjection. We successfully regenerated the injected protoplasts to calli expressing green fluorescent protein (GFP) signals when cultured in optimal medium and cultivation procedures.
Topics: DNA; Microinjections; Polyethylene Glycols; Protoplasts; Transfection
PubMed: 35258834
DOI: 10.1007/978-1-0716-2164-6_14 -
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 -
Methods in Cell Biology 2019Methods for microinjection into sea urchin eggs have become relatively easier because of the technical improvements by a number of researchers in the past decades.... (Review)
Review
Methods for microinjection into sea urchin eggs have become relatively easier because of the technical improvements by a number of researchers in the past decades. However, the size and the characteristics, such as the elasticity and toughness, of the eggs and embryos differ in species, so that we still need to modify the details of methods to adapt to each target. In this section, I list microinjection methods for three species: Hemicentrotus pulcherrimus, which has relatively tough eggs, Temnopleurus reevesii, which has slightly weak eggs, and Strongylocentrotus purpuratus, which is the most used species in sea urchin biology. In addition, I describe the methods for co-injection of morpholino anti-sense oligonucleotides and mRNAs, as well as the method for microinjection into blastomeres.
Topics: Animals; Blastomeres; Embryo, Nonmammalian; Microinjections; Oocytes; Sea Urchins
PubMed: 30777175
DOI: 10.1016/bs.mcb.2018.09.013 -
Journal of Plant Physiology Jan 2020The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern cell biology. Microinjection techniques include the...
The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern cell biology. Microinjection techniques include the penetration of the plasma membrane and, if present, the cell wall with micropipettes, and the application of pressure or electrical currents to drive the micropipette contents into the cell. These procedures interfere with cellular functions and therefore may induce artifacts. We designed the diffusive injection micropipette (DIMP) that avoids most of the possible artifacts due to the drastically reduced volume of its fluid contents and the utilization of diffusion for cargo delivery into the target cell. DIMPs were successfully tested in plant, fungal, and animal cells. Using the continuity of cytoplasmic dynamics over ten minutes after impalement of Nicotiana trichome cells as a criterion for non-invasiveness, we found DIMPs significantly less disruptive than conventional pressure microinjection. The design of DIMPs abolishes major sources of artifacts that cannot be avoided by other microinjection techniques. Moreover, DIMPs are inexpensive, easy to produce, and can be applied without specific equipment other than a micromanipulator. With these features, DIMPs may become the tool of choice for studies that require the least invasive delivery possible of materials into live cells.
Topics: Aspergillus niger; Cell Line; Fluorescent Dyes; Humans; Microinjections; Nicotiana
PubMed: 31765880
DOI: 10.1016/j.jplph.2019.153060