Intratesticular injection followed by electroporation allows gene transfer in caprine spermatogenic cells

Pramod, R. Kumar; Mitra, Abhijit

doi:10.1038/s41598-018-21558-9

Cited by 8 publications

(6 citation statements)

References 58 publications

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“…After a specific interval, injected males can then be used to naturally mate with females to produce transgenic founders. TMGT was initially applied in sheep and goats to produce transgenic founders with inserted genes including lipoprotein lipase (LPL) (Qin et al, 2012), solute carrier family 7 member 11 (SLC7A11) (He et al, 2012), peroxisome proliferator–activated receptor gamma (PPARγ) (Qin et al, 2013), myogenin (MyoG) (Zhang et al, 2014c), and enhanced green fluorescent protein (eGFP) (Raina et al, 2015; Pramod and Mitra, 2018). In general, despite the potential possibilities of sperm-based strategies to mediate transgenesis, these strategies still require optimization.…”

Section: Overview Of Genetic Modification and Relevant Biotechnologicmentioning

confidence: 99%

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

et al. 2019

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Sheep and goats are valuable livestock species that have been raised for their production of meat, milk, fiber, and other by-products. Due to their suitable size, short gestation period, and abundant secretion of milk, sheep and goats have become important model animals in agricultural, pharmaceutical, and biomedical research. Genome engineering has been widely applied to sheep and goat research. Pronuclear injection and somatic cell nuclear transfer represent the two primary procedures for the generation of genetically modified sheep and goats. Further assisted tools have emerged to enhance the efficiency of genetic modification and to simplify the generation of genetically modified founders. These tools include sperm-mediated gene transfer, viral vectors, RNA interference, recombinases, transposons, and endonucleases. Of these tools, the four classes of site-specific endonucleases (meganucleases, ZFNs, TALENs, and CRISPRs) have attracted wide attention due to their DNA double-strand break-inducing role, which enable desired DNA modifications based on the stimulation of native cellular DNA repair mechanisms. Currently, CRISPR systems dominate the field of genome editing. Gene-edited sheep and goats, generated using these tools, provide valuable models for investigations on gene functions, improving animal breeding, producing pharmaceuticals in milk, improving animal disease resistance, recapitulating human diseases, and providing hosts for the growth of human organs. In addition, more promising derivative tools of CRISPR systems have emerged such as base editors which enable the induction of single-base alterations without any requirements for homology-directed repair or DNA donor. These precise editors are helpful for revealing desirable phenotypes and correcting genetic diseases controlled by single bases. This review highlights the advances of genome engineering in sheep and goats over the past four decades with particular emphasis on the application of CRISPR/Cas9 systems.

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Section: Overview Of Genetic Modification and Relevant Biotechnologicmentioning

confidence: 99%

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

et al. 2019

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“…On the other hand, the pIRES2-EGFP after the expression produces into the cell a green-fluorescent protein which stains the transfected cells [28]; the transfection efficiency was evaluated by using the fluorescence microscope Carl Zeiss Axio Vert.A1.…”

Section: Nanoparticle Characterizationmentioning

confidence: 99%

“…Figure 8(a) shows a micrograph of the cells incubated with the AuNP-PEG-pIRES2-EGFP nanoparticles. The transfection study demonstrated that the AuNP-PEG enter into the cells with the pDNA and it allows the expression of the plasmid which stains the cell due to the transcription of the green fluorescent protein [28].…”

Section: Cellular Transfection and Cytotoxicitymentioning

confidence: 99%

Polyethylene Glycol-Coated Gold Nanoparticles as DNA and Atorvastatin Delivery Systems and Cytotoxicity Evaluation

Zamora-Justo

Abrica-González

Vázquez-Martínez

et al. 2019

Journal of Nanomaterials

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The application of nanoscience and nanotechnology in medicine has been useful in the diagnosis, monitoring, and treatment of many diseases. Gold nanoparticles are commonly used for medical imaging studies, biosensors, drug delivery systems, and gene therapy. It has been reported that nanoparticles coated with specific polymers improve the biocompatibility and stability and decrease the cytotoxicity of the nanoparticles. In this work, we performed transfection studies of gold nanoparticles coated with polyethylene glycol, synthetized by two different methods, in a human embryonic kidney cell culture (HEK 293), by using plasmids pSV-β-Gal and pIRES2-EGFP. In addition, we also evaluated the cell uptake of a fluorescent drug (atorvastatin) using the synthetized gold nanoparticles as carriers. Furthermore, the study of cell viability after the interaction between these cells and the nanoparticles was performed. It was shown that the polyethylene glycol-coated gold nanoparticles presented transfection efficiency and cell uptake greater than 45% in each case. These results suggest that the synthetized gold nanoparticles coated with polyethylene glycol could be used successfully and safely as DNA and drug delivery systems.

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“…Electroporation has been employed to transfer genes into various stages of mammalian embryo development, including sperm cells, embryonic stem cells, and preimplantation embryos and post‐implanted fetuses (Chu et al., 1987; Gagne et al., 1991; Osumi & Inoue, 2001; Wall et al., 1992). Pramod and Mitra (2018) reported successful generation of Tg goats by intratesticular injection of a transgene construct (pIRES2‐EGFP) followed by in vivo electroporation of the entire testis. Immunohistochemistry, quantitative real‐time PCR, and western blotting analyses confirmed the expression of GFP in the seminiferous tubules, testicular cells, and subsequent offspring (Pramod & Mitra, 2018).…”

Section: Resultsmentioning

confidence: 99%

Comparison of two methods of sperm‐ and testis‐mediated gene transfer in production of transgenic animals: A systematic review

Dehghan,

Darya,

Mehdinejadiani

et al. 2024

Animal Genetics

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Transgenic (Tg) animal technology is one of the growing areas in biology. Various Tg technologies, each with its own advantages and disadvantages, are available for generating Tg animals. These include zygote microinjection, electroporation, viral infection, embryonic stem cell or spermatogonial stem cell‐mediated production of Tg animals, sperm‐mediated gene transfer (SMGT), and testis‐mediated gene transfer (TMGT). However, there are currently no comprehensive studies comparing SMGT and TMGT methods, selecting appropriate gene delivery carriers (such as nanoparticles and liposomes), and determining the optimal route for gene delivery (SMGT and TMGT) for producing Tg animal. Here we aim to provide a comprehensive assessment comparing SMGT and TMGT methods, and to introduce the best carriers and gene transfer methods to sperm and testis to generate Tg animals in different species. From 2010 to 2022, 47 studies on SMGT and 25 studies on TMGT have been conducted. Mice and rats were the most commonly used species in SMGT and TMGT. Regarding the SMGT approach, nanoparticles, streptolysin‐O, and virus packaging were found to be the best gene transfer methods for generating Tg mice. In the TMGT method, the best gene transfer methods for generating Tg mice and rats were virus packaging, dimethyl sulfoxide, electroporation, and liposome. Our study has shown that the efficiency of producing Tg animals varies depending on the species, gene carrier, and method of gene transfer.

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Intratesticular injection followed by electroporation allows gene transfer in caprine spermatogenic cells

Cited by 8 publications

References 58 publications

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

Polyethylene Glycol-Coated Gold Nanoparticles as DNA and Atorvastatin Delivery Systems and Cytotoxicity Evaluation

Comparison of two methods of sperm‐ and testis‐mediated gene transfer in production of transgenic animals: A systematic review

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