Microinjection Process Research Articles

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot.

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 2 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.

Mosquito Embryo Microinjection under Halocarbon Oil or in Aqueous Solution.

The process of genetically modifying mosquitoes requires skilled delivery of reagents for modification. Plasmids, RNA, DNA, and/or protein must be transported into the developing embryo during an appropriate time in development when these agents will have access to the genome. Embryo microinjection has been the main method by which such modifying agents have been delivered. Ideally the microinjection process will deliver these modifying agents in sufficient quantity to effect the genetic modification without severely damaging or killing the injected embryo in the process. As semiaquatic insects, mosquitoes have embryos that are susceptible to desiccation and the degree to which embryos are susceptible is based on species. Two microinjection methods are outlined here. The first method describes embryo microinjections performed under Halocarbon-27 oil. The oil is used to reduce desiccation during the injection process. A second method limits desiccation by injecting the mosquito embryos in water. In both procedures, the embryos are first aligned and then injected before the embryos cellularize, ∼1 h and 45 min after oviposition.

A simple method to dramatically increase C. elegans germline microinjection efficiency

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.

Micromanipulation System Capable of Simultaneously Presenting High-Resolution and Large Field-of-View Images in Real-Time

Microinjection technology is widely used in biotechnological processes such as gene manipulation and microinsemination. Generally, microinjection is performed under an optical microscope while viewing the video of the targets in real-time. In the microinjection process, multiple oocytes must be placed in the same droplet, for which the injection must be performed multiple times. Furthermore, the target must be observed at different magnifications during injection and the oocyte transfer operation. Hence, the operators are required to change the magnification and light intensity repeatedly. These tasks are complicated and are a burden for the operators. Thus, the accuracy, reproducibility, and productivity of microinjection depend on the operator’s skill level. In this study, to reduce the burden on the operator and simplify the operation of microinjection, we propose a micromanipulation system that enables both wide-range and high-resolution video presentation with free viewpoint selection. The proposed micromanipulation system is based on a view-expansion microscope system with simultaneous multi-view imaging. We verify the effectiveness of the proposed system by evaluating the resolution of images presented to the operator and through the experiments involving moving microbeads performed by inexperienced subjects.

Micro-manipulation Interface that Presents Surface Force Tactile Sensation According to the Cell Deformation

The microinjection technique is widely applied in fields such as biology, medicine, and intracytoplasmic sperm injection (ICSI). Microinjection process requires special skill and, thus, generally ICSI is conducted by embryologists. However, the shortage of embryologists has become a problem in clinics, and there is a need for an easy injection system that does not rely on embryologists. In this study, we propose a micromanipulation interface that assists to perceive cell extension for easy injection system. The proposed interface provides 2-dimensional force/tactile feedback through a fabric actuator with air pressure based on real-time visual sensing of cell deformation. The effectiveness of the proposed interface is verified through puncturing experiments on perceiving cell extension perfumed by inexperienced subjects.

Design and Development of an Automatic Microinjection System for High-Throughput Injection of Zebrafish Larvae

The microinjection of zebrafish larvae is a complicated work due to their small size and shape variations. In this article, for the first time, we design an automatic microinjection system dedicated to high-throughput injection of zebrafish larvae. The proposed custom-built microarray Petri dish combined with machine vision-based motion control enables high-efficiency automatic identification and injection of a batch of zebrafish larvae. The feasibility of the developed system is demonstrated by experimental testing on live zebrafish larvae. Results show that the developed system works fast with more stable injection success rate and higher survival rate than the skilled experimenter. The proposed system offers a good working efficiency and maintains a consistent success rate and stability in the process of high-throughput injection. It can be applied in various biomedical experiments based on zebrafish microinjection. Note to Practitioners—Zebrafish is widely used in biomedical experiments due to its obvious advantages. At present, the batch injection task of larvae is mainly conducted by experimenters manually. As the number of injections increases, experimenters will get tired, which makes it difficult for manual injection to maintain a stable success rate. This article reports the first automatic microinjection system for high-throughput batch injection of zebrafish larvae. A new microarray Petri dish has been proposed to fix the larvae, facilitating the microinjection process. Experimental results show that the system can complete a large number of injection tasks quickly while ensuring a stable success rate and survival rate of the larvae. It can free experimenters from repeated and tedious injection work. It also enables efficient zebrafish larvae microinjection for inexperienced experimenters.

Evaluation of CRISPR gene-editing tools in zebrafish

BackgroundZebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create ‘knockout’ models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies.MethodsTo systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression.ResultsWe compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (< 1%). To characterize if commonly used ‘mock’ CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the ‘mock’ injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation.ConclusionOverall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.

Effect of poly(ε-caprolactone) microfibers in poly(lactide-co-glycolide) based bone fixation plate on preventing dimensional shrinkage and promoting cell interactions

Owing to numerous merits, Poly(lactide-co-glycolide) (PLGA) based materials exhibit great potential in orthopedic application. However, the inherent low stiffness, poor dimensional stability and weak cell interaction are still obstacles to fulfill the role in practical application. In this study, we reported a facile production of PLGA/Poly(ε-caprolactone) (PCL) bone fixation plate with enhanced mechanical and cell adhesion properties by the micro-injection process. The in-situ fibrillation of PCL at high injection rate constructed unique parent-daughter fibril morphology, which promoted the interaction of the interfaces between the PLGA and PCL. Impressively, the tensile modulus of the as-prepared fixation plate increased from 25.64 MPa to 98.08 MPa and maintained its original dimension after 40 days immersion in aqueous conditions. The well oriented PCL fibrils with high crystallinity served as barrier in prevention of the slip PLGA molecular chain as well as the invasion of water molecules, which played a key role in promoting cell proliferation. The comprehensive properties of PLGA/PCL fixation plate not only pave a new way to enhance the short-term cell attachment/proliferation in PLGA-based materials, but also inspire the designing principle for the modification of future bone tissue engineering products.

Microinjection System to Enable Real-Time 3D Image Presentation Through Focal Position Adjustment

The microinjection technique, in which DNA is injected into cells, is widely applied in fields such as biology, medicine, and pharmacy. However, the implementation of the microinjection process requires skill and, thus, it is necessary to improve the operability. To this end, this study proposes a microinjection system that improves depth visibility through real-time 3D image presentation. An algorithm to estimate 3D position and size of micro-targets using a focal position adjustment mechanism is implemented to the system. In addition, a calibration method to adjust relative position and orientation between a target object and micromanipulators is developed. Then, the system presents reconstructed 3D images of a target and micromanipulators. Finally, the effectiveness of the proposed system is verified through manipulation experiments involving porcine embryos.

Molecular dynamics simulation on the adhesion mechanism at polymer‐mold interface of microinjection molding

AbstractIn the demolding process of microinjection molding, the defects of microstructures are often caused by the strong adhesion between polymer and mold. In order to study the adhesion mechanism, the molecular dynamics (MD) method was proposed to simulate the adsorption of cycloolefin copolymer (COC) molecules on mold surfaces. The evolution snapshots of COC molecular chains of three interfacial models were obtained to directly demonstrate the adhesive strength of interfaces. Meanwhile, the work of adhesion, the relative concentration, the potential energy, and the radial distribution function (RDF) were calculated to explain the interaction mechanism of polymer‐mold interfaces. The simulation results showed that the COC‐Ni interface had the largest work of adhesion and the lowest potential energy, compared with other two interfaces. The van der Waals (VDW) energy, which mainly derived from the interaction between H atoms in COC and the mold material was the only nonbond interaction energy at the COC‐Ni and COC‐Si interfaces, while the electrostatic energy existed in COC‐Al2O3 interface. In order to reduce the adhesion between polymer and mold, fluorine (F) element could be doped into the Ni mold.

Precise Cell Injection and Extraction Control Based on Microscopic Visual Feedback

Nuclear transplantation is an effective method for processing, modifying, and reconstructing cells, which is widely used in biological experiments. Microinjections and extractions are key steps in nuclear transplantation. It is beneficial to the promotion and the application of nuclear transplantation by increasing the efficiency and improving the quality of microinjections. At present, little research has been implemented for the speed/volume control problem in the microinjection process. Motivated by this observation, this article designs an update-law-improved adaptive control scheme to implement precise injections and extractions. Specifically, a precise mathematical model of the overall system is established first, which takes the imaging, pneumatic pump, cell, and micropipette elements into full consideration. Since parameters in the model are difficult to calibrate or measure, an update-law-improved adaptive control algorithm, together with a trajectory planner, is constructed for the microinjection and extraction system, which shows great tolerance to parameter uncertainties, avoids overshoot of the system to ensure great protection for the manipulated cells, and demonstrates fast convergence rate. The stability of the designed control system is proved theoretically by LaSalle’s principle. Finally, the controller is tested by both numerical simulation and practical experiments, with the obtained data demonstrating satisfactory performances of the overall system.

Automated manipulation of zebrafish embryos using an electrothermal microgripper

We present the first report on automated micro-manipulation of zebrafish embryos using an electrothermally-actuated microgripper. A five-bar linkage compliant microgripper, driven by a V-shaped electrothermal micro-actuator, is designed based on topological optimization. The electrothermally-actuated microgripper is fabricated, tested, and then integrated into a robotic micromanipulation system. The semi-automated manipulation of a single zebrafish embryo, as well as a series of comparison experiments, is carried out. Experimental results demonstrate that the microgripper has reliable capability of picking, moving, holding and releasing the zebrafish embryo. Specifically, it performs fairly well in immobilizing and limiting the excessive deformation of the embryo. Finally, based on teaching, the whole process of zebrafish embryo microinjection is programed and performed automatically, thus demonstrating great strengths and performance of the microgripper in zebrafish embryo micro-manipulation.

A Survey of Force-Assisted Robotic Cell Microinjection Technologies

Cell injection plays an important role in genetics, transgenics, molecular biology, drug discovery, reproductive study, and other biomedical fields. Compared with manual cell microinjection and robotic cell microinjection with sole position feedback, force-assisted robotic cell microinjection can improve the success rate and survival rate of cell injection. In this paper, the state-of-the-art research on microinjection of both adherent cells and suspended cells with microforce sensing techniques is reviewed. The significance of force sensors in the robotic cell injection system is also discussed. Five types of prevalent force sensing methods and their applications in cell microinjection are reviewed. The challenges and promising solutions in automating the cell microinjection process are addressed. Note to Practitioners —Microinjection process is a complex task to perform. As the advance progress of high-performance microforce sensors, force-assisted robotic cell injection has been a hot topic in recent years. This paper presents the state-of-the-art survey of recent developments on microforce sensing for robotic cell microinjection to address the research challenges. Based on microforce sensing and control, microinjection of both adherent and suspended cells can benefit from the force-assisted robotic cell injection process. The main challenges and promising solutions in terms of micromanipulator design, injection control design, cell holder design, penetration scheme design, injecting pipette maintenance, injection volume, cost reduction, and microforce sensor calibration issues have been discussed. The related research trends are summarized.

Study on optogenetics technology and the design of its script for virtual simulation experiment

Optogenetics is one of the biggest breakthroughs in neurobiology in recent decades, which is a revolutionary approach to precise intervention of specific cell functions through the combination of optical and genetic engineering techniques. However, this technique involves the interdisciplinary theoretical knowledge and methods, and it requires expensive equipments and long experimental period. Thus, it is not convenient to carry out widely in experiment teaching for undergraduates except the virtual simulation teaching. In present virtual simulation experiment, we aim to learn the basic principle, operation and application of optogenetics and achieve the desired teaching effects by simulating the process of the virus package, simulating the process of the animal selection, surgery and virus stereotactic microinjection, simulating the expression process of photosensitive channel, the extracellular verification process of photosensitive channel function in vivo, and simulating the observation process that optogenetic inhibition of glutamate neurons in the amygdala affect animal fear behavior. In this paper, the content design of the script making of the virtual experiment has been discussed in the above ways. Key words: Virtual simulation experiment; Experimental teaching; Optogenetics; Script

Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis

A mechanistic understanding of evolutionary developmental biology requires the development of novel techniques for the manipulation of gene function in phylogenetically diverse organismal systems. Recently, gene-specific knockdown by microinjection of short hairpin RNA (shRNA) was applied in the sea anemone Nematostella vectensis, demonstrating that the shRNA approach can be used for efficient and robust sequence-specific knockdown of a gene of interest. However, the time- and labor-intensive process of microinjection limits access to this technique and its application in large scale experiments. To address this issue, here we present an electroporation protocol for shRNA delivery into Nematostella eggs. This method leverages the speed and simplicity of electroporation, enabling users to manipulate gene expression in hundreds of eggs or embryos within minutes. We provide a detailed description of the experimental procedure, including reagents, electroporation conditions, preparation of Nematostella eggs, and follow-up care of experimental animals. Finally, we demonstrate the knockdown of several endogenous and exogenous genes with known phenotypes and discuss the potential applications of this method.

Force-Sensing Robotic Microinjection System for Automated Multi-Cell Injection With Consistent Quality

This paper presents the design and development of a new microinjection system with force sensing for automated multi-cell injection in genome editing of zebrafish using the CRISPR/Cas9 system. In comparison with the traditional approach, it achieves a high injection speed and high precision of operation, which leads to consistent quality and high survival rate of the injected cells. One unique feature is that force sensing is employed to determine the exact moment when the cell membrane is pierced, which contributes to the protection of the cell from damage. Moreover, a new cell holding device is designed to immobilize the cells, which speeds up the multi-cell microinjection process by eliminating the use of conventional vacuum holding pipette. The system implements the object detection by machine vision algorithm along with position/force regulation. The machine vision is realized by combining the pyramid template matching algorithm with Kalman filter, which reduces the computation time and facilitates real-time control. The experimental studies are conducted to verify the performance of the developed system. Results demonstrate the reliability of the robotic microinjection system, which offers a more consistent injection quality and higher viability of cells over the skilled human operator.

A Review of Automated Microinjection of Zebrafish Embryos.

Cell microinjection is a technique of precise delivery of substances into cells and is widely used for studying cell transfection, signaling pathways, and organelle functions. Microinjection of the embryos of zebrafish, the third most important animal model, has become a very useful technique in bioscience. However, factors such as the small cell size, high cell deformation tendency, and transparent zebrafish embryo membrane make the microinjection process difficult. Furthermore, this process has strict, specific requirements, such as chorion softening, avoiding contacting the first polar body, and high-precision detection. Therefore, highly accurate control and detection platforms are critical for achieving the automated microinjection of zebrafish embryos. This article reviews the latest technologies and methods used in the automated microinjection of zebrafish embryos and provides a detailed description of the current developments and applications of robotic microinjection systems. The review covers key areas related to automated embryo injection, including cell searching and location, cell position and posture adjustment, microscopic visual servoing control, sensors, actuators, puncturing mechanisms, and microinjection.

Production of Transgenic Mice by Pronuclear Microinjection.

Pronuclear microinjection remains the most widely used method for the germline modification of mice and other species. The method is conceptually quite simple and at least in rodents can produce transgenic offspring with relatively high efficiency. Here, we describe the various components of the production of transgenic mice including a detailed list of materials and equipment. We outline in detail the preparation of animals, the retrieval, culture and transfer of embryos, the preparation of DNA, and the microinjection process. We have added a substantial collection of notes with helpful suggestions that reflect the many years of experience we have using this technology and our continuing efforts to improve animal welfare and the efficiency of producing transgenics.

Влияние процесса микроинъекции на выживаемость эмбрионов зебрафиш (Danio rerio) при получении трансгенных рыб

Влияние процесса микроинъекции на выживаемость эмбрионов зебрафиш (Danio rerio) при получении трансгенных рыб

Evaluation of mold roughness influence on injected thin micro-cavities

Due to its ability to produce low-cost and high repeatable micro-polymeric parts, micro-injection molding is emerging as one of the most promising enabling technologies in many different fields. When approaching the micro-scale, different issues related to the process should be addressed, especially as the thickness of the cavities becomes very thin. In particular, the mold roughness affects the surface quality of the micro-components produced, like in macro-molding, but this could also encompass the complete filling of the parts. The main objective of the present paper is to evaluate the roughness influence of the mold on the non-Newtonian fluid flow during the micro-injection process in thin micro-cavities where the mold surface condition could affect the process due to the phenomena neglected in macro- or mesoscale as less important boundary conditions. The part design was properly conceived in order to neglect the effect of dimensions and geometry and to highlight the roughness contribution during the filling phase. The experimentation was performed using cavities with different roughness values and decreasing depths from 200 to 50 μm, considerably lower than 250 μm found in literature and manufactured by the micro-electrical discharge machining (μ-EDM). The results of the experiments are discussed and show that cavity-surface roughness affects the process as the molding scale is decreased. In particular, in thin cavities, higher surface roughness promotes the filling of component.