Microinjection Method Research Articles

An Effective Microinjection Method and TALEN-Mediated Genome Editing in Pacific Abalone.

Pacific abalone, Haliotis discus hannai, is an economically important marine mollusk species and an important model animal for studies on ecological, fertilization and developmental biology. While embryonic injection and genome editing have been wildly used in gene function study and trait improvement in many species, they have not been developed in abalones. In this study, we reported an effective method to inject exogenous materials in H. discus hannai unfertilized eggs. The injected eggs could be fertilized at a ratio of 52.6% ± 5.9% and hatch at a ratio of 14.6% ± 1.6%. On the base of this, we further developed an efficient genome editing approach in this species with the transcription activator-like effector nuclease (TALEN) technique. Two TALEN pairs targeting the coding sequence of the abalone nodal gene were assembled and tested. While one of the TALEN pairs showed no detectable mutation efficacy, the other one generated mutations in 50% of the targeted loci. The mutation includes small insertions and deletions and base pair replacements like that reported in other species when the TALEN method was applied. Overall, this is the first study to demonstrate site-specific genome editing in abalone. This work can serve as a reference for future studies focusing on the functional genomics in mollusks.

A Microinjection Method for Infecting the Planthopper Sogatella furcifera (Hemiptera: Delphacidae) with the Southern Rice Black-Streaked Dwarf Virus.

The southern rice black-streaked dwarf virus (SRBSDV) causes significant economic damage to rice crops. This virus is transmitted to rice plants by the planthopper Sogatella furcifera (Horváth) in a persistent, circular, and propagative manner. Researchers currently lack suitable methods for assaying the activity of SRBSDV in vitro and preserving the virus all year. We used a microinjection method to directly inject SRBSDV extracts into the hemocoel of S. furcifera nymphs. SRBSDV was subsequently detected by Reverse Transcription-Polymerase Chain Reaction in more than 56.7% of the insects after 5 d and 60% of healthy rice plants fed by these insects also became SRBSDV infected. Moreover, injecting planthopper with an extract of SRBSDV-infected rice plant that had been frozen at -80°C for 220 d caused 63.3% to become viruliferous. These results indicate that SRBSDV can be successfully transmitted to S. furcifera by microinjection, and that extracts of SRBSDV-infected rice plants frozen at -80°C for 220 d still contain sufficient active SRBSDV to infect S. furcifera. We provide a novel way to preserve SRBSDV all year by injecting S. furcifera with the SRBSDV extract.

Microinjection methods for sea urchin eggs and blastomeres.

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.

Advanced microinjection protocol for gene manipulation using the model newt Pleurodeles waltl.

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.

An effective microinjection method for genome editing of marine aquaculture fish: tiger pufferfish Takifugu rubripes and red sea bream Pagrus major

Genome editing technology is becoming increasingly accepted as a way to improve traits in marine fish aquaculture. In fish, microinjection is a major method for introducing RNA or protein into eggs for genome editing; however, this method has not yet been established in aquaculture fish. We successfully established microinjection methods achieving high survival rates for tiger pufferfish and red sea bream by optimizing the following three parameters: (1) the soaking solution of fertilized eggs during microinjection, (2) the elapsed time from in vitro fertilization to microinjection, (3) the elapsed time from stripping to microinjection. In tiger pufferfish, Iwamatsu solution or diluted sea water is effective as the soaking solution. In vitro fertilization can be performed at intervals of 15 min from fertilization until 2.5 h after stripping. Similarly, in red sea bream, Leibovitz’s L-15 medium or Iwamatsu solution is effective as the soaking solution and in vitro fertilization can be performed at intervals of 10 min from fertilization until 2.5 h after stripping. We anticipate our findings will contribute to effectively establish genome edited aquaculture breeds.

Influence of Heterologous Transplant of DNA-lacking Mitochondria from Entamoeba histolytica on Proliferation of Entamoeba invadens.

In mitochondria, compatibility of proteins encoded in mitochondrial DNA and nuclear DNA is essential for the normal functioning of the organelle. Incompatibility between mitochondrial and nuclear DNA can lead to dysfunctional respiration, mitochondrial diseases, and lethal problems, which suggests that the presence of heterologous mitochondria is unfavorable. In a previous study, we established a transplant method for DNA-lacking mitochondria (mitosomes) in the anaerobic protozoan Entamoeba histolytica. In this study, interspecies transplant of mitosomes from E.histolytica into Entamoeba invadens, which is a parasitic protozoon of reptiles, was performed using the microinjection method at various temperatures and injection volumes. When E.invadens was used as recipient, it showed higher tolerance to a lower temperature and larger injection volume, in comparison with E.histolytica. After microinjection, donor mitosomes expressing HA-tag conjugated protein were observed in recipient cells by immunofluorescent staining. The heterologous mitosomes-injected cells proliferated and growth rate of the microinjected-cells was similar to that of intact cells. Therefore, we conclude that interspecies transplant of DNA-lacking mitochondria does not result in incompatibility.

The Emergence of the Tardigrade Hypsibius exemplaris as a Model System.

The success of scientists in revealing biological mechanisms has depended in large part on choosing tractable model systems. In 1997, molecular phylogenetics revealed that two of biology's most tractable models-Caenorhabditis elegans and Drosophila-are much more closely related to each other than had been thought previously. I began to explore whether any of the little-studied members of this branch of the tree of life might serve as a new model for comparative biology that could make use of the rich and ongoing sources of information flowing from C. elegans and Drosophila research. Tardigrades, also known as water bears, make up a phylum of microscopic animals. The tardigrade Hypsibius exemplaris (recently disambiguated from a closely related species, Hypsibius dujardini) can be maintained in laboratories and has a generation time of <2 wk at room temperature. Stocks of animals can be stored frozen and revived. The animals and their embryos are optically clear, and embryos are laid in groups, with each synchronous clutch of embryos laid in a clear molt. We have developed techniques for laboratory study of this system, including methods for microinjection of animals, immunolocalization, in situ hybridization, RNA interference, transcriptomics, and methods for identifying proteins that mediate tolerance to extreme environments. Here, I review the development of this animal as an emerging model system, as well as recent molecular studies aimed at understanding the evolution of developmental mechanisms that underpin the evolution of animal form and at understanding how biological materials can survive extreme environments.

Production of Transgenic Bovine Embryos by Microinjection Method of a Lentiviral Vector in Zygotes

Objective: To produce bovine transgenic embryos by microinjection of a lentiviral vector that carries the eGFP gene as a marker in zygotes six hours after fertilization. Methods: 834 oocytes were matured and subjected to one of four treatments designed as follows: CC: Control: IVF with Cumulus-oocyte with (COCs), cultivated in CR2 medium supplemented with 10% FBS and incubated at 38.5°C in atmosphere of 95% humidity and 5% CO₂.CCM: Control culture medium: fertilized in vitro for six hours, cultured in medium SOF aluminum in pouches under the same conditions of CC. MC: Microinjection control: Fertilized under the same treatment conditions CCM. After six hours they were microinjected with TALP medium and cultured in sachets with the same conditions of CCM treatment. ML: Microinjected with the lentivirus: Fertilized in the same conditions of the CCM treatment. After six hours they were microinjected with the lentiviral vector carrying the eGFP transgene and cultured in sachets with the same treatment conditions CCM and MC. Findings: The cleavage rate found in CC was higher (p 0.05) in them, but yes, with ML (p < 0.05). On average, 76.4% of the zygotes obtained in ML expressed the green fluorescent protein. Application/Improvements: The cult ure conditions used were suitable for CC, CCM and MC, microinjection with lentiviral vector has some influence on embryo development, it succeeded in obtaining transgenic zygotes. Keywords: Green Fluorescent Protein (eGFP), Genetic Modification, Micromanipulation of Zygotes

Identification of RNAi-related genes and transgenerational efficiency of RNAi in Artemia franciscana

Artemia and the RNA interference technique were used in research to explore the function of genes in crustaceans. In this study, we annotated and characterized nine putative genes involved in RNA interference in Artemia franciscana including two Dicer, three Argonauts, two dsRNA binding proteins, Drosha and Sid-1 together with evidence of Pasha and Exportin-5. The phylogenetic results indicated the identity of these genes and revealed that genes homologous to those in both the siRNA and miRNA pathway in arthropods are also present in Artemia franciscana. This study provides the first look at the core genes of the RNAi machinery of A. franciscana. Additionally, egg-sac microinjection was used to generate the desired RNAi phenotype. The high numbers of nauplii with the desired phenotypes obtained from injected maternal A. franciscana (both from the first and second brood) demonstrated the reliability of the egg-sac microinjection method, providing a methodology to study gene function on high numbers of individuals be treating the parental generation.

Easy and efficient production of completely embryonic-stem-cell-derived mice using a micro-aggregation device.

There is an increasing demand for genetically modified mice produced without crossing, for rapid phenotypic screening studies at the organismal level. For this purpose, generation of completely embryonic-stem-cell (ESC)-derived chimeric mice without crossing is now possible using a microinjection or aggregation method with 3i culture medium. However, the microinjection of ESCs into blastocyst, morula, or 8-cell-stage embryos requires a highly skilled operator. The aggregation method is an easier alternative, but the conventional aggregation protocol still requires special skills. To make the aggregation method easier and more precise, here we developed a micro-aggregation device. Unlike conventional 3-dimensional culture, which uses hanging-drop devices for aggregation, we fabricated a polystyrene funnel-like structure to smoothly drop ESCs into a small area (300-μm in diameter) at the bottom of the device. The bottom area was designed so that the surface tension of the liquid-air interface prevents the cells from falling. After aggregation, the cells can be recovered by simply exerting pressure on the liquid from the top. The microdevice can be set upon a regular 96-well plate, so it is compatible with multichannel pipette use or machine operation. Using the microdevice, we successfully obtained chimeric blastocysts, which when transplanted resulted in completely ESC-derived chimeric mice with high efficiency. By changing the number of ESCs in the aggregate, we found that the optimum number of co-cultured ESCs was around 90~120 per embryo. Under this condition, the efficiency of generating completely ESC-derived mice was the same or better than that of the injection method. These results indicated that our microdevice can be used to produce completely ESC-derived chimeric mice easily and with a high success rate, and thus represents a promising alternative to the conventional microinjection or aggregation method, especially for high-throughput, parallel experimental applications.

Establishment of a method for Lutzomyia longipalpis sand fly egg microinjection: The first step towards potential novel control strategies for leishmaniasis

Leishmaniasis is a vector-borne parasitic disease transmitted by sand flies that affects 1.3 million people across 98 countries, with limited control strategies due to the lack of an available vaccine and the emergence of insecticide resistance. Novel control strategies that are being explored for mosquito-borne diseases, such as Wolbachia bacterial inhibition of pathogens and genetically modified insects (e.g. using CRISPR-Cas9 editing), rely on the ability to consistently inject embryos of the target species. Here we present a novel method to obtain and inject preblastoderm sand fly embryos of the genus Lutzomyia (Lu.)longipalpis, the principle vector of zoonotic visceral leishmaniasis in South America. The procedures required to obtain sufficiently young Lu. longipalpis colony embryos are described alongside a microinjection technique that permits rapid injection and minimal handling of small sand fly embryos post-injection. Using a strain of Wolbachia as a 'marker' for successful injection, our protocol produced early generation Wolbachia transinfected Lu. longipalpis lines, demonstrating its potential as the first step for use in novel applied strategies for sand fly control.

Keratin 4 regulates the development of human white sponge nevus.

The aim of this study was to investigate the roles of keratin 4 (KRT4) gene in the development of human white sponge nevus (WSN). Transgenic mice were created using the microinjection method with pcDNA3.1 vectors expressing KRT4 wild-type (WT) gene and E520K mutation. Polymerase chain reaction (PCR) and Western blotting were used to identify the genotype of transgenic founders and their filial generations. Expression of KRT4 in mouse oral mucosa was characterized by immunohistochemistry (IHC), and the whole epithelium layer of transgenic mice was observed using transmission electron microscope (TEM). The positive rate of KRT4 transgenic mice in F1 generation was 45.5%. Expression level of KRT4 protein was significantly higher in 2-month-old transgenic mice than WT mice. Furthermore, all the epithelial lamina of 3-month-old transgenic mice showed reduced staining of KRT4. The surface and spinous layers were full of hyalocytes and bubble cells, which are similar to the clinical symptoms of WSN. For the ultrastructure, both tonofilaments and Odland bodies increased. Our study indicated the mutated KRT4 gene may play important roles in the pathogenesis of WSN.

Establishment of a method for Lutzomyia longipalpis sand fly embryo microinjection: The first step towards potential novel control strategies for leishmaniasis

Leishmaniasis is a vector-borne parasitic disease transmitted by sand flies that affects 1.3 million people across 98 countries, with limited control strategies due to the lack of an available vaccine and the emergence of insecticide resistance. Novel control strategies that are being explored for mosquito-borne diseases, such as Wolbachia bacterial inhibition of pathogens and genetically modified insects (e.g. using CRISPR-Cas9 editing), rely on the ability to consistently inject embryos of the target species. Here we present a novel method to obtain and inject preblastoderm sand fly embryos of the genus Lutzomyia (Lu.) longipalpis, the principle vector of zoonotic visceral leishmaniasis in South America. The procedures required to obtain sufficiently young Lu. longipalpis colony embryos are described alongside a microinjection technique that permits rapid injection and minimal handling of small sand fly embryos post-injection. Using a strain of Wolbachia as a ‘marker’ for successful injection, our protocol produced early generation Wolbachia transinfected Lu. longipalpis lines, demonstrating its potential as the first step for use in novel applied strategies for sand fly control.

I-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases

We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.

Creating a mouse model resistant to induced ischemic stroke and cardiovascular damage

Vascular prostanoids, isomerized from an intermediate prostaglandin (PG), H2, produced by cyclooxygenase (COX), exert various effects on the vascular system, both protective and destructive. During endothelial dysfunction, vascular protector prostacyclin/prostaglandin I2 (PGI2) is decreased, while inflammatory PGE2 and thrombotic TXA2 are increased. Therefore, our research aim was to reverse the event by controlling PGH2 metabolism by generating an in vivo model via enzymatic engineering of COX-1 and prostacyclin synthase (PGIS). The COX-1 and PGIS genes were linked to a 10-residue amino acid linker to form a Single-chain Enzyme Complex (SCHEC), COX-1-10aa-PGIS. Transgenic (CP-Tg) mice in a FVB/N background were generated using the pronuclear microinjection method. We first confirmed mRNA and protein expression of COX-1-10aa-PGIS in various CP-Tg mouse tissues, as well as upregulation of circulating PGI2. We then examined the cardiovascular function of these mice. Our CP-Tg mice exhibited marked resistance to vascular assault through induced carotid arterial blockage, acute thrombotic stroke and arterial arrest, angiotensin-induced peripheral vasoconstriction, and hepatic lipid accumulation after receiving a high-fat diet. They also had a longer lifespan compared with wild-type mice. This study raises the possibility of fighting cardiovascular diseases by regulating cellular arachidonic acid-derived PGH2 metabolites using enzymatic engineering.

Transplantation of Spermatogonial Stem Cells Suspension into Rete Testis of Azoospermia Mouse Model.

The loss of spermatogonia following chemo-or radiotherapy leading to temporary or permanent infertility of the patient is a well known and unwanted side effect of many oncological therapies. In this study, germ cells were isolated from 4 days old mouse testis cells. Busulfan treatment was used to the eliminate proliferating cells in the testis of recipient mice. The donor cells suspended in DMEM, were introduced into the rete testis of recipient mice via microinjection method. To distinguish the progeny of the transplanted donor stem cells from endogenous germ cells, BrdU-labeled cells were used. In addition, real time PCR was performed to determine expression levels of ngn3 and LIN28 (spermatogonia stem cells markers)before and after transplantation. Western blot analysis was further performed to detect an increase in - ngn3 expression after transplantation. Transplantations of stem cells into rete testis of the recipients was done. Our results clearly showed a significant increase in spermatozoa number in epididymal luman Spermatogonial stem cells (SSCs) did not show alkaline phosphatase activities while ngn3 and LIN28 were clearly expressed. Ngn3 and LIN28 expression were reduced after busulfan treatment compared to untreatmented mice. However, the expression of ngn3 and LIN28 increased after transplantation . BrdU-labeled testis cells were successfully transplanted into rete testis of recipient mice. These cells remained in rete testis of all recipient mice up to two months after transplantation. The present study clearly confirme that a regeneration after cytotoxic treatment was based on morphological criteria. We demonstrated the increase in stem cell numbers during regeneration and after transplantation. Transplantation of spermatogonial stem cells suspension by the injection of cells via the rete testis of recipient azoospermia model considerably enhances the efficiency of this procedure.

Practical Guide for Ascidian Microinjection: Phallusia mammillata.

Phallusia mammillata has recently emerged as a new ascidian model. Its unique characteristics, including the optical transparency of eggs and embryos and efficient translation of exogenously introduced mRNA in eggs, make the Phallusia system suitable for fluorescent protein (FP)-based imaging approaches. In addition, genomic and transcriptomic resources are readily available for this ascidian species, facilitating functional gene studies. Microinjection is probably the most versatile technique for introducing exogenous molecules such as plasmids, mRNAs, and proteins into ascidian eggs/embryos. However, it is not practiced widely within the community; presumably, because the system is rather laborious to set up and it requires practice. Here, we describe in as much detail as possible two microinjection methods that we use daily in the laboratory: one based on an inverted microscope and the other on a stereomicroscope. Along the stepwise description of system setup and injection procedure, we provide practical tips in the hope that this chapter might be a useful guide for introducing or improving a microinjection setup.

Electrical-assisted microinjection for analysis of fertilization and cell division in mammalian oocytes and early embryos.

Microinjection is an essential approach in the study of mammalian oocytes and early embryos, and is useful for the introduction of many molecules and reagents. Whereas microinjection into germinal vesicle stage oocytes is relatively simple using various microinjection setups, metaphase-II mouse eggs are notoriously fragile, and nondamaging microinjection can be difficult to achieve. Here we describe a microinjection method that is based on electrophysiology, which vastly reduces microinjection damage, especially in metaphase-II eggs. When optimized, this approach allows for over 90% oocyte survival, increasing confidence in experimental results.

Microinjection of Exogenous Nucleic Acids into Eggs: Ciona Species.

Microinjection is a common technique used to deliver nucleic acids into eggs and embryos in Ciona species. There are three Ciona species that are commonly used for research-Ciona intestinalis type A (C. robusta), C. intestinalis type B (C. intestinalis), and C. savignyi. Here, we present the microinjection methods using eggs and embryos of C. intestinalis type A and C. savignyi; however, our methods would also be applicable to eggs and embryos of C. intestinalis type B. Microinjection is a classical and widely used delivery method, which involves the use of a glass micropipette, a hollow glass needle with a microscopic tip, to inject nucleic acids into eggs and embryos under a stereo microscope. The required amount of nucleic acids is much smaller for microinjection than for electroporation, another delivery method. Proteins, and other chemicals, such as fluorescent dye, can be introduced with nucleic acids using a microinjection.

Artificial Infection of Ticks with Borrelia burgdorferi Using a Microinjection Method and Their Detection In Vivo Using Quantitative PCR Targeting flaB RNA.

Borrelia burgdorferi is maintained in nature by a tick-rodent infection cycle where it traverses and colonizes a variety of host and vector tissues. A tick-borne murine model has been developed to study Lyme disease in the laboratory, which has a substantial impact in advancing our knowledge of spirochete infectivity and pathogenesis. Here, we detail a microinjection-based method for rapid and efficient infection of ticks with B. burgdorferi. While laboratory generation of B. burgdorferi-infected nymphs via natural larval engorgement on infected hosts and subsequent molting could take several weeks to months, the microinjection-based infection procedure requires only a few hours to generate infected ticks and allows introduction of defined quantities of spirochetes, including mutant isolates that are attenuated for infection in mice and thus cannot be naturally acquired by ticks. We also describe a quantitative PCR-based protocol for the measurement of B. burgdorferi in tick and murine hosts targeting spirochete RNA that is highly efficient, reproducible, and a better surrogate of active infection.