Functional primordial germ cell–like cells from pluripotent stem cells in rats

Science, Год журнала: 2022, Номер 376(6589), С. 176 - 179

Опубликована: Апрель 7, 2022

The in vitro generation of germ cells from pluripotent stem (PSCs) can have a substantial effect on future reproductive medicine and animal breeding. A decade ago, gametogenesis was established the mouse. However, induction primordial cell-like (PGCLCs) to produce gametes has not been achieved any other species. Here, we demonstrate functional PGCLCs rat PSCs. We show that epiblast-like floating aggregates form PGCLCs. gonadal somatic support maturation epigenetic reprogramming When are transplanted into seminiferous tubules germline-less rats, spermatids-that is, those capable siring viable offspring-are generated. Insights our model will elucidate conserved divergent mechanisms essential for broad applicability gametogenesis.

Язык: Английский

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Principles of Genetic Engineering

Genes, Год журнала: 2020, Номер 11(3), С. 291 - 291

Опубликована: Март 10, 2020

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety approaches. For example, homologous recombination can be used target specific sequences mouse embryonic stem (ES) cell genomes or other cultured cells, but it cumbersome, poorly efficient, and relies on drug positive/negative selection culture for success. Other routinely applied methods include random integration after direct transfection (microinjection), transposon-mediated insertion, insertion mediated by viral vectors production transgenic mice rats. Random occurs more frequently than recombination, has numerous drawbacks, despite its efficiency. The most elegant effective method based guided endonucleases, because these sequences. Since advent clustered regularly interspaced short palindromic repeats CRISPR/Cas9 technology, endonuclease-mediated gene targeting become widely engineer supplanting zinc finger nucleases, transcription activator-like effector meganucleases. Future improvements editing may achieved increasing efficiency homology-directed repair. Here, we describe principles genetic detail: (1) how common elements current technologies need chromosome break occur, (2) sensitive genotyping assays detect altered (3) delivery modalities that impact characterization modifications. In summary, while some remain steadfast, others change as are ever-evolving continue revolutionize research many fields.

Язык: Английский

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Skin graft with dermis and appendages generated in vivo by cell competition

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Апрель 29, 2024

Autologous skin grafting is a standard treatment for defects such as burns. No artificial substitutes are functionally equivalent to autologous grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of epidermal cells on synthetic dermal substitute, can wounds, it requires wound bed be well-vascularized appendages. In this study, we successfully generate complete grafts with pluripotent stem cell-derived appendages p63 knockout embryos' dermis. Donor keratinocytes encroach by eliminating based cell-extracellular matrix adhesion mediated cell competition. chimeric contains allogenic dermis, engraftable long Furthermore, could semi-humanized segments human injection into amnionic cavity mice embryos. Niche encroachment opens possibility graft production in livestock animals.

Язык: Английский

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Generation of Functional Organs Using a Cell-Competitive Niche in Intra- and Inter-species Rodent Chimeras

Cell stem cell, Год журнала: 2020, Номер 28(1), С. 141 - 149.e3

Опубликована: Дек. 28, 2020

Язык: Английский

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Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models

Frontiers in Genetics, Год журнала: 2021, Номер 12

Опубликована: Апрель 20, 2021

The rat has been extensively used as a small animal model. Many genetically engineered models have emerged in the last two decades, and advent of gene-specific nucleases accelerated their generation recent years. This review covers techniques advances to generate lines application development more broadly, such conditional knockouts reporter gene strains. In addition, genome-editing that remain be explored are discussed. also focuses particularly on areas which extensive work done: human genetic diseases immune system analysis. Models thoroughly described these highlight competitive advantages over available corresponding mouse versions. objective this is provide comprehensive description potential for addressing specific scientific questions characterize best genome-engineering tools developing new projects.

Язык: Английский

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Electroporation-Mediated Genome Editing of Livestock Zygotes

Frontiers in Genetics, Год журнала: 2021, Номер 12

Опубликована: Апрель 13, 2021

The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level skill. Electroporation offers simplified more streamlined approach to transfect zygotes. There are number studies examining the parameters used in electroporation mouse rat Here, we review conditions, timing, success rates that have reported for mice rats, addition few reports about livestock zygotes, specifically pigs cattle. at, soon after, fertilization can help reduce rate mosaicism, presence two genotypes cells an individual; as nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic large species with long generation intervals it take years obtain non-mosaic, homozygous offspring through breeding. Gene knockouts via non-homologous end joining pathway widely successfully using gene knock-ins. Delivering DNA plasmids zygote hindered zona pellucida (ZP), majority knock-ins short single stranded (ssDNA) repair templates, typically less 1 kb. most promising deliver larger donor templates up 4.9 kb along without injection, use recombinant adeno-associated viruses (rAAVs) combination electroporation. However, similar other methods clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this also associated levels mosaicism. Recent developments complementing germline ablated individuals edited germline-competent offer avoid mosaicism founder lines. Even electroporation-mediated delivery there remain additional chokepoints pipeline currently hinder scalable production non-mosaic livestock.

Язык: Английский

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Pre-Clinical Models in Implant Dentistry: Past, Present, Future

Biomedicines, Год журнала: 2021, Номер 9(11), С. 1538 - 1538

Опубликована: Окт. 26, 2021

Biomedical research seeks to generate experimental results for translation clinical settings. In order improve the transition from bench bedside, researchers must draw justifiable conclusions based on data an appropriate model. Animal testing, as a prerequisite human exposure, is performed in range of species, laboratory mice larger animals (such dogs or non-human primates). Minipigs appear be animal choice studying bone surgery around intraoral dental implants. Dog models, well-known field implant research, tend now used studies conducted under compromised oral conditions (biofilm). Regarding small mostly use rodents, with interest rabbit models declining. Mouse remain reference genetic studies. On other hand, over last decade, scientific advances and government guidelines have led replacement, reduction, refinement all research. new development strategies, some vivo experiments are being progressively replaced by vitro biomaterial approaches. this review, we summarize key information currently available highlight (i) pros cons each type, (ii) levels decisional procedures regarding study objectives, (iii) outlook discussing possible non-animal options.

Язык: Английский

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Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice

BMC Biology, Год журнала: 2024, Номер 22(1)

Опубликована: Фев. 2, 2024

Abstract Background The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led the use this delivery method for production genetically altered knock-in mice via CRISPR-Cas9. potential exists simplify and extend types alleles that can be generated directly in zygote, obviating need manipulations genome embryonic stem cell route. Results We present data from a total 13 models using CRISPR-Cas9 electroporation zygotes donor repair templates transduction with virus. explore efficiency gene targeting at 12 independent genetic loci effects allele complexity introduce strategies efficient identification founder animals. In addition, we investigate reliability germline transmission engineered methodology. By comparing our against cells their microinjection into blastocysts, assess animal cost two methods. Conclusions Our results confirm provides robust effective route mice, across range insertion sizes (0.9–4.7 kb). find is considerably less than generating thus constitutes considerable 3Rs reduction.

Язык: Английский

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Genome engineering with Cas9 and AAV repair templates, successes and pitfalls

Mammalian Genome, Год журнала: 2025, Номер unknown

Опубликована: Янв. 13, 2025

Genome editing, in particular the CRISPR/Cas9 system, is widely used to generate new animal models. However, generation of mutations, such as conditional knock-out or knock-in, can remain complex and inefficient, because difficulty deliver donor DNA (single double stranded) into nucleus fertilized oocytes. The use recombinant adeno-associated viruses (rAAV) a rapidly developing approach that promises improve efficiency creation In this mini-review, we explore progress challenges using combination with rAAV for precise genome editing. We will summarise current knowledge transduction, data on its rodent embryos easily sequence replacements insertions, limitations unexpected events observed date, protocol optimisations already place facilitate

Язык: Английский

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Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Cells, Год журнала: 2020, Номер 9(4), С. 799 - 799

Опубликована: Март 26, 2020

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune are considered an important tool assessing the function of genes interest various biological systems. Thus, development efficient and simple methods to produce genome-edited (GE) animals would accelerate research this field. CRISPR/Cas9 system was initially employed early embryos, utilizing classical gene delivery such microinjection or electroporation, which required ex vivo handling zygotes before transfer recipients. Recently, novel genome editing via oviductal nucleic acid (GONAD), improved GONAD (i-GONAD), transplacental for acquiring fetuses (TPGD-GEF), facilitate easy embryo manipulation, have been established. Studies these techniques pregnant female mice direct introduction genome-editing components into oviduct were dependent on tail-vein injection. In mice, embryogenesis occurs within oviducts uterus, often hampers genetic manipulation especially those at postimplantation stages (days 6 8), owing a thick surrounding layer tissue called decidua. review, we surveyed recent achievements production GE outlined advantages disadvantages process. We also referred past stage embryos germ cells primordial spermatogonial stem cells, will benefit relevant research.

Язык: Английский

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