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STAR Protocols Sep 2021In targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of germline editing is attributed to the syncytial nature...
In targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in , to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).
Topics: Animals; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Gene Editing; Genetic Engineering; Microinjections
PubMed: 34505086
DOI: 10.1016/j.xpro.2021.100748 -
International Journal of Molecular... Oct 2019Oil palm (, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands... (Review)
Review
Oil palm (, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.
Topics: Agrobacterium tumefaciens; Arecaceae; CRISPR-Cas Systems; Gene Editing; Microinjections; Palm Oil; Plant Somatic Embryogenesis Techniques; Polyethylene Glycols; Protoplasts; Tissue Culture Techniques; Transformation, Genetic
PubMed: 31661801
DOI: 10.3390/ijms20215353 -
Journal of Visualized Experiments : JoVE Mar 2023The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order...
The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders. As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.
Topics: Animals; Humans; Macaca mulatta; Callithrix; Microinjections; Brain; Induced Pluripotent Stem Cells; Electroporation; Organoids; Mammals
PubMed: 37036224
DOI: 10.3791/65176 -
Trends in Biotechnology Sep 2018Genetic engineering of plants has enhanced crop productivity in the face of climate change and a growing global population by conferring desirable genetic traits to... (Review)
Review
Genetic engineering of plants has enhanced crop productivity in the face of climate change and a growing global population by conferring desirable genetic traits to agricultural crops. Efficient genetic transformation in plants remains a challenge due to the cell wall, a barrier to exogenous biomolecule delivery. Conventional delivery methods are inefficient, damaging to tissue, or are only effective in a limited number of plant species. Nanoparticles are promising materials for biomolecule delivery, owing to their ability to traverse plant cell walls without external force and highly tunable physicochemical properties for diverse cargo conjugation and broad host range applicability. With the advent of engineered nuclease biotechnologies, we discuss the potential of nanoparticles as an optimal platform to deliver biomolecules to plants for genetic engineering.
Topics: Agrobacterium tumefaciens; Biolistics; Cell Wall; Crops, Agricultural; Electroporation; Gene Editing; Genetic Engineering; Genome, Plant; Government Regulation; Humans; Microinjections; Nanoparticles; Plant Cells; Plants, Genetically Modified; Transformation, Genetic; Transgenes
PubMed: 29703583
DOI: 10.1016/j.tibtech.2018.03.009 -
Archivum Immunologiae Et Therapiae... Apr 2015Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high... (Review)
Review
Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.
Topics: Animals; Animals, Genetically Modified; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cattle; Fibroblasts; Humans; Immunoglobulin Constant Regions; Immunotherapy; Mice; Microinjections; Oocytes; Protein Engineering; Rats; Species Specificity
PubMed: 25467949
DOI: 10.1007/s00005-014-0322-x -
Journal of Plastic, Reconstructive &... Aug 2022Mesenchymal stem cell (MSC)-supplemented acellular nerve allografts (ANA) are a potential strategy to improve the treatment of segmental nerve defects. Prior to clinical...
BACKGROUND
Mesenchymal stem cell (MSC)-supplemented acellular nerve allografts (ANA) are a potential strategy to improve the treatment of segmental nerve defects. Prior to clinical translation, optimal cell delivery methods must be defined. While two techniques, dynamic seeding and microinjection, have been described, the seeding efficiency, cell viability, and distribution of MSCs in ANAs are yet to be compared.
METHODS
Sciatic nerve segments of Sprague-Dawley rats were decellularized, and MSCs were harvested from the adipose tissue of Lewis rats. Cell viability was evaluated after injection of MSCs through a 27-gauge needle at different flow rates (10, 5, and 1 µL/min). MSCs were dynamically seeded or longitudinally injected into ANAs. Cell viability, seeding efficiency, and distribution were evaluated using LIVE/DEAD and MTS assays, scanning electron microscopy, and Hoechst staining.
RESULTS
No statistically significant difference in cell viability after injection at different flow rates was seen. After cell delivery, 84.1 ± 3.7% and 87.8 ± 2.8% of MSCs remained viable in the dynamic seeding and microinjection group, respectively (p = 0.41). The seeding efficiency of microinjection (100.4%±5.6) was significantly higher than dynamic seeding (48.1%±8.6) on day 1 (p = 0.001). Dynamic seeding demonstrated a significantly more uniform cell distribution over the course of the ANA compared to microinjection (p = 0.02).
CONCLUSION
MSCs remain viable after both dynamic seeding and microinjection in ANAs. Higher seeding efficiency was observed with microinjection, but dynamic seeding resulted in a more uniform distribution. In vivo studies are required to assess the effect on gene expression profiles and functional motor outcomes.
Topics: Allografts; Animals; Mesenchymal Stem Cells; Microinjections; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley
PubMed: 35570113
DOI: 10.1016/j.bjps.2022.04.017 -
Advanced Drug Delivery Reviews Mar 2018Conventional systematically-administered drugs distribute evenly throughout the body, get degraded and excreted rapidly while crossing many biological barriers, leaving... (Review)
Review
Conventional systematically-administered drugs distribute evenly throughout the body, get degraded and excreted rapidly while crossing many biological barriers, leaving minimum amounts of the drugs at pathological sites. Controlled drug delivery aims to deliver drugs to the target sites at desired rates and time, thus enhancing the drug efficacy, pharmacokinetics, and bioavailability while maintaining minimal side effects. Due to a number of unique advantages of the recent microfluidic lab-on-a-chip technology, microfluidic lab-on-a-chip has provided unprecedented opportunities for controlled drug delivery. Drugs can be efficiently delivered to the target sites at desired rates in a well-controlled manner by microfluidic platforms via integration, implantation, localization, automation, and precise control of various microdevice parameters. These features accordingly make reproducible, on-demand, and tunable drug delivery become feasible. On-demand self-tuning dynamic drug delivery systems have shown great potential for personalized drug delivery. This review presents an overview of recent advances in controlled drug delivery using microfluidic platforms. The review first briefly introduces microfabrication techniques of microfluidic platforms, followed by detailed descriptions of numerous microfluidic drug delivery systems that have significantly advanced the field of controlled drug delivery. Those microfluidic systems can be separated into four major categories, namely drug carrier-free micro-reservoir-based drug delivery systems, highly integrated carrier-free microfluidic lab-on-a-chip systems, drug carrier-integrated microfluidic systems, and microneedles. Microneedles can be further categorized into five different types, i.e. solid, porous, hollow, coated, and biodegradable microneedles, for controlled transdermal drug delivery. At the end, we discuss current limitations and future prospects of microfluidic platforms for controlled drug delivery.
Topics: Drug Delivery Systems; Humans; Microfluidic Analytical Techniques; Microinjections; Needles
PubMed: 28919029
DOI: 10.1016/j.addr.2017.09.013 -
Scientific Reports Apr 2021In recent years, animals and plants have received increasing attention as potential next-generation protein production systems, especially for biopharmaceuticals and...
In recent years, animals and plants have received increasing attention as potential next-generation protein production systems, especially for biopharmaceuticals and animal proteins. The aim of the present study was to develop the earthworms Eisenia fetida Waki and Eisenia andrei Sagami as next-generation animal protein production hosts. These earthworms have been approved as model animals for acute toxicity tests by the Organization for Economic Co-operation and Development, and they have post-translational modification systems. However, so far, none of the studies have used earthworm transfection techniques. Thus, we developed a transfection method for E. fetida and E. andrei using microinjection and electroporation systems. The maximum survival rates and transfection efficiencies were 79.2% and 29.2% for E. fetida, and 95.8% and 50.0% for E. andrei, respectively. Furthermore, human erythropoietin was detected in the transformed earthworm tail fragments using an enzyme-linked immunosorbent assay. These results contribute to the development of a potential earthworm-based novel animal protein production system.
Topics: Animals; Electroporation; Erythropoietin; Gene Expression; Humans; Microinjections; Models, Animal; Oligochaeta; Protein Engineering; Transfection
PubMed: 33854163
DOI: 10.1038/s41598-021-87641-w -
Drug Delivery and Translational Research Aug 2015In the literature, several types of microneedles have been extensively described. However, porous microneedle arrays only received minimal attention. Hence, only little... (Review)
Review
In the literature, several types of microneedles have been extensively described. However, porous microneedle arrays only received minimal attention. Hence, only little is known about drug delivery via these microneedles. However, porous microneedle arrays may have potential for future microneedle-based drug and vaccine delivery and could be a valuable addition to the other microneedle-based drug delivery approaches. To gain more insight into porous microneedle technologies, the scientific and patent literature is reviewed, and we focus on the possibilities and constraints of porous microneedle technologies for dermal drug delivery. Furthermore, we show preliminary data with commercially available porous microneedles and describe future directions in this field of research.
Topics: Drug Delivery Systems; Equipment Design; Humans; Microinjections; Nanopores; Needles; Permeability; Pharmaceutical Preparations; Skin; Vaccines
PubMed: 26044672
DOI: 10.1007/s13346-015-0238-y -
Methods in Cell Biology 2020The study of mitosis has always relied on bulk-preparation biochemistry techniques (Mazia & Dan, 1952), but very early on lent itself to living, single cell microscopic...
The study of mitosis has always relied on bulk-preparation biochemistry techniques (Mazia & Dan, 1952), but very early on lent itself to living, single cell microscopic techniques (Inoue, 1953; Taylor, 1959). Here we describe several of the methods used by our lab to study cell division in living cultured cells, including cold-induced mitotic arrest, cold-induced chromosome missegregation, same-cell live and fixed cell imaging, and microinjection of inactivating antibodies. We detail our imaging system based on an upright fluorescent microscope and spinning disk confocal, as well as the customized "HEKS" metal support slide imaging chambers.
Topics: Animals; Cell Culture Techniques; Cell Line; Chlorocebus aethiops; Humans; Imaging, Three-Dimensional; Mammals; Microinjections; Mitosis; Rats; Research; Spindle Apparatus
PubMed: 32423650
DOI: 10.1016/bs.mcb.2020.02.001