RESUMO
Previous studies on paternal epigenetic inheritance have shown that sperm RNAs play a role in this type of inheritance. The microinjection of sperm small noncoding RNAs into fertilised mouse oocytes induces reprogramming of the early embryo, which is thought to be responsible for the differences observed in adult phenotype. While sperm long noncoding RNAs (lncRNAs) have also been investigated in a previous study, their microinjection into fertilised oocytes did not yield conclusive results regarding their role in modulating brain development and adult behavioural phenotypes. Therefore, in the current study we sought to investigate this further. We used our previously established paternal corticosterone (stress hormone) model to assess sperm lncRNA expression using CaptureSeq, a sequencing technique that is more sensitive than the ones used in other studies in the field. Paternal corticosterone exposure led to dysregulation of sperm long noncoding RNA expression, which encompassed lncRNAs, circular RNAs and transposable element transcripts. Although they have limited functional annotation, bioinformatic approaches indicated the potential of these lncRNAs in regulating brain development and function. We then separated and isolated the sperm lncRNAs and performed microinjections into fertilised oocytes, to generate embryos with modulated lncRNA populations. We observed that the resulting adult offspring had lower body weight and altered anxiety and affective behavioural responses, demonstrating roles for lncRNAs in modulating development and brain function. This study provides novel insights into the roles of lncRNAs in epigenetic inheritance, including impacts on brain development and behaviours of relevance to affective disorders.
Assuntos
Corticosterona , Microinjeções , RNA Longo não Codificante , Espermatozoides , Animais , Masculino , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , Camundongos , Corticosterona/farmacologia , Espermatozoides/metabolismo , Microinjeções/métodos , Feminino , Epigênese Genética , Camundongos Endogâmicos C57BL , Ansiedade/metabolismo , Ansiedade/genética , Oócitos/metabolismo , Comportamento Animal/fisiologia , Estresse Psicológico/metabolismo , Encéfalo/metabolismoRESUMO
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.
Assuntos
Agulhas , Humanos , Sistemas de Liberação de Medicamentos , Animais , Nanomedicina Teranóstica , Microinjeções/instrumentação , Microinjeções/métodosRESUMO
Microneedles are a transdermal drug delivery system in which the needle punctures the epithelium to deliver the drug directly to deep tissues, thus avoiding the influence of the first-pass effect of the gastrointestinal tract and minimizing the likelihood of pain induction. Hydrogel microneedles are microneedles prepared from hydrogels that have good biocompatibility, controllable mechanical properties, and controllable drug release and can be modified to achieve environmental control of drug release in vivo. The large epithelial tissue in the oral cavity is an ideal site for drug delivery via microneedles. Hydrogel microneedles can overcome mucosal hindrances to delivering drugs to deep tissues; this prevents humidity and a highly dynamic environment in the oral cavity from influencing the efficacy of the drugs and enables them to obtain better therapeutic effects. This article analyzes the materials and advantages of common hydrogel microneedles and reviews the application of hydrogel microneedles in the oral cavity.
Assuntos
Sistemas de Liberação de Medicamentos , Hidrogéis , Boca , Agulhas , Hidrogéis/química , Humanos , Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Animais , Microinjeções/instrumentação , Microinjeções/métodosRESUMO
The various kinds of nanocarriers (NCs) have been explored for the delivery of therapeutics designed for the management of skin manifestations. The NCs are considered as one of the promising approaches for the skin delivery of therapeutics attributable to sustained release and enhanced skin penetration. Despite the extensive applications of the NCs, the challenges in their delivery via skin barrier (majorly stratum corneum) have persisted. To overcome all the challenges associated with the delivery of NCs, the microneedle (MN) technology has emerged as a beacon of hope. Programmable drug release, being painless, and its minimally invasive nature make it an intriguing strategy to circumvent the multiple challenges associated with the various drug delivery systems. The integration of positive traits of NCs and MNs boosts therapeutic effectiveness by evading stratum corneum, facilitating the delivery of NCs through the skin and enhancing their targeted delivery. This review discusses the barrier function of skin, the importance of MNs, the types of MNs, and the superiority of NC-loaded MNs. We highlighted the applications of NC-integrated MNs for the management of various skin ailments, combinational drug delivery, active targeting, in vivo imaging, and as theranostics. The clinical trials, patent portfolio, and marketed products of drug/NC-integrated MNs are covered. Finally, regulatory hurdles toward benchtop-to-bedside translation, along with promising prospects needed to scale up NC-integrated MN technology, have been deliberated. The current review is anticipated to deliver thoughtful visions to researchers, clinicians, and formulation scientists for the successful development of the MN-technology-based product by carefully optimizing all the formulation variables.
Assuntos
Administração Cutânea , Sistemas de Liberação de Medicamentos , Agulhas , Dermatopatias , Pele , Humanos , Sistemas de Liberação de Medicamentos/métodos , Dermatopatias/tratamento farmacológico , Pele/metabolismo , Pele/efeitos dos fármacos , Nanopartículas/química , Nanopartículas/administração & dosagem , Portadores de Fármacos/química , Animais , Absorção Cutânea , Microinjeções/métodos , Microinjeções/instrumentaçãoRESUMO
BACKGROUND: Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene. METHODS AND RESULTS: Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp. CONCLUSIONS: This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Microinjeções , Mutação , Triticum , Triticum/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Microinjeções/métodos , Mutação/genética , Pólen/genéticaRESUMO
Microneedle (MN) technology has been extensively studied for its advantages of minimal invasiveness and user-friendliness. Notably, hydrogel microneedles (HMNs) have garnered considerable attention for biofluid extraction due to its high swelling properties and biocompatibility. This review provides a comprehensive overview of definition, materials, and fabrication methods associated with HMNs. The extraction mechanisms and optimization strategies for enhancing extraction efficiency are summarized. Moreover, particular emphasis is placed on HMN-based biofluid extraction and detection in the domains of food and agriculture, encompassing the detection of small molecules, nucleic acids, and other relevant analytes. Finally, current challenges and possible solutions associated with HMN-based biofluid extraction are discussed.
Assuntos
Técnicas Biossensoriais , Hidrogéis , Sistemas de Liberação de Medicamentos/métodos , Agulhas , Microinjeções/métodos , AgriculturaRESUMO
Microneedles are a promising micro-scale drug delivery platform that has been under development for over two decades. While 3D printing technology has been applied to fabricate these systems, the challenge of achieving needle sharpness remains. In this study, we present an innovative approach for microneedle fabrication using digital light processing (DLP) 3D printing and smart chitosan biomaterial. For the first time, we used hydroxybutyl methacrylated chitosan (HBCMA), which possesses dual temperature- and photo-sensitive properties, to create microneedles. The DLP approach enabled a quick generation of HBCMA-based microneedles with a high resolution. The microneedles exhibited 4D properties with a change in needle dimensions upon exposure to temperature, which enhances resolution, sharpens needles, and improves mechanical strength. We demonstrated the ability of these microneedles to load, deliver, sustained release small molecular drugs and penetrate soft tissue. Overall, the HBCMA-based microneedles show promising potential in non-dermal drug delivery applications.
Assuntos
Quitosana , Administração Cutânea , Microinjeções/métodos , Sistemas de Liberação de Medicamentos/métodos , Preparações de Ação RetardadaRESUMO
Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor-intensive preparatory steps. Here, we constructed a machine-vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4× increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in 2â days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms.
Assuntos
Robótica , Animais , Humanos , Peixe-Zebra/genética , Microinjeções/métodos , Drosophila melanogaster/genética , Animais Geneticamente ModificadosRESUMO
Microneedles (MNs) are micron-sized needles, typically <2 mm in length, arranged either as an array or as single needle. These MNs offer a minimally invasive approach to ocular drug delivery due to their micron size (reducing tissue damage compared to that of hypodermic needles) and overcoming significant barriers in drug administration. While various types of MNs have been extensively researched, significant progress has been made in the use of hollow MNs (HMNs) for ocular drug delivery, specifically through suprachoroidal injections. The suprachoroidal space, situated between the sclera and choroid, has been targeted using optical coherence tomography-guided injections of HMNs for the treatment of uveitis. Unlike other MNs, HMNs can deliver larger volumes of formulations to the eye. This review primarily focuses on the use of HMNs in ocular drug delivery and explores their ocular anatomy and the distribution of formulations following potential HMN administration routes. Additionally, this review focuses on the influence of formulation characteristics (e.g., solution viscosity, particle size), HMN properties (e.g., bore or lumen diameter, MN length), and routes of administration (e.g., periocular transscleral, suprachoroidal, intravitreal) on the ocular distribution of drugs. Overall, this paper highlights the distinctive properties of HMNs, which make them a promising technology for improving drug delivery efficiency, precision, and patient outcomes in the treatment of ocular diseases.
Assuntos
Administração Oftálmica , Sistemas de Liberação de Medicamentos , Olho , Agulhas , Humanos , Animais , Olho/metabolismo , Preparações Farmacêuticas/administração & dosagem , Microinjeções/métodos , Microinjeções/instrumentaçãoRESUMO
OBJECTIVE: The objective of the present study was to enhance the bioavailability of cannabidiol (CBD) using 3D Digital Light Processing (DLP)-printed microneedle (MN) transdermal drug delivery system. METHODS: CBD MN patch was fabricated and optimized using 3D DLP printing using CBD (8% w/v), Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (0.49% w/v), distilled water (20% w/v), and poly (ethylene glycol) dimethacrylate 550 (PEGDAMA 550) (up to 100% w/v). CBD MNs were characterized for their morphology, mechanical strength, in vitro release study, ex vivo permeation study, and in vivo pharmacokinetic (PK) profile. KEY FINDINGS: Microscopic images showed that sharp CBD MNs with a height of ~800 µm, base diameter of ~250 µm, and tip with a radius of curvature (RoC) of ~15 µm were successfully printed using optimized printing parameters. Mechanical strength studies showed no significant deformation in the morphology of CBD MNs even after applying 0.5N/needle force. Ex vivo permeation study showed significant (P < .0001) permeation of CBD in the receiving media as compared to CBD patch (control). In vivo PK study showed significantly (P < .05) enhanced bioavailability in the case of CBD MN patch as compared to CBD subcutaneous inj. (control). CONCLUSION: Overall, systemic absorption of CBD was significantly enhanced using 3D-printed MN drug delivery system.
Assuntos
Administração Cutânea , Disponibilidade Biológica , Canabidiol , Sistemas de Liberação de Medicamentos , Agulhas , Impressão Tridimensional , Adesivo Transdérmico , Animais , Canabidiol/farmacocinética , Canabidiol/administração & dosagem , Ratos , Masculino , Absorção Cutânea , Ratos Sprague-Dawley , Microinjeções/métodos , Liberação Controlada de FármacosRESUMO
Microneedles (MNs) offer minimally-invasive access to interstitial fluid (ISF) - a potent alternative to blood in terms of monitoring physiological analytes. This property is particularly advantageous for the painless detection and monitoring of drugs and biomolecules. However, the complexity of the skin environment, coupled with the inherent nature of the analytes being detected and the inherent physical properties of MNs, pose challenges when conducting physiological monitoring using this fluid. In this review, we discuss different sensing mechanisms and highlight advancements in monitoring different targets, with a particular focus on drug monitoring. We further list the current challenges facing the field and conclude by discussing aspects of MN design which serve to enhance their performance when monitoring different classes of analytes.
Assuntos
Microinjeções , Agulhas , Animais , Humanos , Técnicas Biossensoriais/métodos , Monitoramento de Medicamentos/métodos , Líquido Extracelular/metabolismo , Microinjeções/instrumentação , Microinjeções/métodos , Pele/metabolismoRESUMO
Porous Microneedles (PMNs) have been widely used in drug delivery and medical diagnosis owing to their abundant interconnected pores. However, the mechanical strength, the use of organic solvent, and drug loading capacity have long been challenging. Herein, a novel strategy of PMNs fabrication based on the Ice Templating Method is proposed that is suitable for insoluble, soluble, and nanosystem drug loading. The preparation process simplifies the traditional microneedle preparation process with a shorter preparation time. It endows the highly tunable porous morphology, enhanced mechanical strength, and rapid dissolution performance. Micro-CT three-dimensional reconstruction was used to better quantify the internal structures of PMNs, and we further established the equivalent pore network model to statistically analyze the internal pore structure parameters of PMNs. In particular, the mechanical strength is mainly negatively correlated with the surface porosity, while the dissolution velocity is mainly positively correlated with the permeability coefficient by the correlation heatmap. The poorly water-soluble Asiatic acid was encapsulated in PMNs in nanostructured lipid carriers, showing prominent hypertrophic scar healing trends. This work offers a quick and easy way of preparation that may be used to expand PMNs function and be introduced in industrial manufacturing development.
Assuntos
Sistemas de Liberação de Medicamentos , Agulhas , Solubilidade , Porosidade , Gelo , Liberação Controlada de Fármacos , Animais , Microinjeções/métodos , Microinjeções/instrumentação , Microtomografia por Raio-X , Portadores de Fármacos/química , Lipídeos/químicaRESUMO
Microneedles (MN) have emerged as an innovative technology for drug delivery, offering a minimally invasive approach to administer therapeutic agents. Recent applications have included ocular drug delivery, requiring the manufacture of sub-millimeter needle arrays in a reproducible and reliable manner. The development of 3D printing technologies has facilitated the fabrication of MN via mold production, although there is a paucity of information available regarding how the printing parameters may influence crucial issues such as sharpness and penetration efficacy. In this study, we have developed and optimized a 3D-printed MN micro-mold using stereolithography (SLA) 3D printing to prepare a dissolving ocular MN patch. The effects of a range of parameters including aspect ratio, layer thickness, length, mold shape and printing orientation have been examined with regard to both architecture and printing accuracy of the MN micro-mold, while the effects of printing angle on needle fidelity was also examined for a range of basic shapes (conical, pyramidal and triangular pyramidal). Mechanical strength and in vitro penetration of the polymeric (PVP/PVA) MN patch produced from reverse molds fabricated using MN with a range of shapes and height, and aspect ratios were assessed, followed by ex vivo studies of penetration into excised scleral and corneal tissues. The optimization process identified the parameters required to produce MN with the sharpest tips and highest dimensional fidelity, while the ex vivo studies indicated that these optimized systems would penetrate the ocular tissue with minimal applied pressure, thereby allowing ease of patient self-administration.
Assuntos
Administração Oftálmica , Sistemas de Liberação de Medicamentos , Agulhas , Impressão Tridimensional , Estereolitografia , Animais , Microinjeções/métodos , Microinjeções/instrumentação , Córnea/metabolismo , Esclera , Suínos , Tecnologia Farmacêutica/métodosRESUMO
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.
Assuntos
Agulhas , Polímeros , Humanos , Polímeros/química , Sistemas de Liberação de Medicamentos/instrumentação , Animais , Microinjeções/instrumentação , Microinjeções/métodos , Monitorização Fisiológica/instrumentação , Monitorização Fisiológica/métodosRESUMO
For successful treatment of diseases, sufficient therapeutics must be provided to the body. Microneedle applications in therapeutic delivery and analytics sampling are restricted because of various issues, including smaller area for drug loading and analytics sampling. To achieve sufficient drug loading and analytics sampling and improve drug penetration while maintaining painless administration, patch-type microneedle arrays were designed and fabricated using polymer casting from a conical cavity mold. Microcavities were formed on a carbon plate via micromechanical machining. A porous polymer layer was coated on a microneedle patch (MNP). The pores of the porous polymer layer provided space and channels for drug delivery. A pH-sensitive polymer layer was employed to cap the porous polymer layer, which prevented drug leakage during storage and provided a stimulus drug release in response to body pH conditions. The drug can be delivered through holes connected to both sides of the patch. The drug release of the MNP was investigated in vitro and in vivo and showed conceptual proof that these MNs have the potential to enhance treatment protocols for various diseases with the flexibility of coating and therapeutic materials and offer significant scope for further variations and advancement.
Assuntos
Carbono , Sistemas de Liberação de Medicamentos , Agulhas , Sistemas de Liberação de Medicamentos/métodos , Carbono/química , Animais , Liberação Controlada de Fármacos , Microinjeções/instrumentação , Microinjeções/métodos , Porosidade , Concentração de Íons de Hidrogênio , Polímeros/química , CamundongosRESUMO
Precise integration of DNA constructs greater than 3 kb into mouse zygotes is difficult. Here, we present a protocol for large DNA transgenesis in mice using the Cas9+Bxb1 toolbox. We describe steps for choosing mouse strains with preplaced attachment sites. We then detail procedures for microinjecting mouse zygotes with the plasmid donor DNA construct to generate transgenic mice by recombination-mediated cassette exchange. This protocol has the potential for application in exploring the functional implications of large structural variations in cancer. For complete details on the use and execution of this protocol, please refer to Low et al.1 and Hosur et al.2.
Assuntos
DNA , Técnicas de Transferência de Genes , Camundongos Transgênicos , Animais , Camundongos , DNA/genética , Sistemas CRISPR-Cas/genética , Zigoto/metabolismo , Microinjeções/métodos , Plasmídeos/genética , FemininoRESUMO
Neurological disorders, including brain injury, brain tumors, and neurodegenerative diseases, rank as the second leading cause of death worldwide. Exploring effective new treatments for neurological disorders has long been a hot research issue in clinical practice. Recently, microneedles (MNs) have attracted much attention due to their designation as a "painless and non-invasive" novel transdermal delivery method, characterized by their biocompatibility and sustainability. The advantages of MNs open an avenue for potential therapeutic interventions targeting neurological disorders. This review presents a concise overview of progress in the field of MNs, with highlights on the application in the treatment of neurological disorders. Notably, trends in the development of MNs and future challenges are also discussed.
Assuntos
Administração Cutânea , Sistemas de Liberação de Medicamentos , Microinjeções , Agulhas , Doenças do Sistema Nervoso , Humanos , Sistemas de Liberação de Medicamentos/métodos , Doenças do Sistema Nervoso/tratamento farmacológico , Animais , Microinjeções/métodosRESUMO
In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.
Assuntos
Sistemas CRISPR-Cas , Microinjeções , Robótica , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/embriologia , Sistemas CRISPR-Cas/genética , Microinjeções/métodos , Robótica/métodos , Interferência de RNA , Embrião não Mamífero , Técnicas de Silenciamento de Genes/métodos , Proteínas de Peixe-Zebra/genética , RNA Mensageiro/genéticaRESUMO
Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.
Assuntos
Drosophila , Técnicas de Transferência de Genes , Microinjeções , Animais , Microinjeções/métodos , Técnicas de Transferência de Genes/instrumentação , Drosophila/genética , Drosophila/embriologia , Plasmídeos/genética , Plasmídeos/administração & dosagem , Embrião não Mamífero , Animais Geneticamente Modificados , Integrases/genéticaRESUMO
During development, the zebrafish embryo relies on its yolk sac as a nutrient source. Here, we present a protocol for modifying the free fatty acid (FFA) and triacylglycerol (TAG) content of the zebrafish yolk sac by microinjection. We describe steps for needle and injection mold preparation, FFA and TAG solution preparation, and microinjection. This protocol can elucidate how excesses of FFA and TAG affect development and modify the transcriptome of zebrafish embryos. For complete details on the use and execution of this protocol, please refer to Konadu et al. 1.