Production Of Chimeras Research Articles

Unreduced spore formation in a spontaneous chimeric pinnule in an artificially produced haploid Anisocampium niponicum (Athyriaceae, Polypodiales).

Haploid sporophytes of Anisocampium niponicum with 2n = 40, were produced artificially by induced apogamy in vitro. They were subsequently transplanted into pots and two of them have been cultivated for the investigation of sporogenesis and/or production of chimera for more than 20years. Haploid A. niponicum is sterile, but an abnormal chimeric pinnule that developed spontaneously in a single frond produced sporangia with spores. Each sporangium bore approximately 32 spores that were almost uniform in size. Sowing of these spores resulted in 50 gametophytes. Of 20 gametophytes cultured individually, five produced sporophytes apogamously after eight months. Both the gametophytes and subsequent apogamous sporophytes showed a chromosome number of 2n = 40. Our study demonstrates that a haploid sporophyte offspring can be produced from a haploid mother sporophyte via haploid spores. Since asexual reproduction is a prominent evolutionary process in ferns, the reproduction of a haploid A. niponicum sporophyte by unreduced spore formation might help to elucidate how apogamous ferns occur and evolve.

Recovering a Targeted Mutation in Mice from Embryonic Stem Cell Chimeras or CRISPR-Cas Founders.

Following the production of chimeras from targeted embryonic stem (ES) cells or obtaining founders from CRISPR-Cas gene editing in preimplantation embryos, the desired targeted mutation must be recovered and established in the heterozygous state in a strain or stock of mice for further study. The breeding schemes for ES chimeras and CRISPR-Cas founders differ. For ES cell chimeras, we discuss the relative benefits of breeding from male or female chimeras. We discuss the importance of genetic background and provide practical advice for putting the mutation on inbred or outbred backgrounds or producing a coisogenic strain. For CRISPR-Cas founders, which will most likely be mosaic for different mutations, initial breeding strategies are discussed to maintain a desired genetic background at the same time as producing progeny to segregate different alleles. Strategies for testing the progeny to recognize indels, missense mutations, and knock-in mutations are discussed. In the event that ES cell chimeras or CRISPR-Cas founders produce no offspring or fail to transmit the mutant allele(s), there is a troubleshooting guide to pinpoint the problem. If heterozygous offspring from the chimeras or founders are normal, fertile, and of both sexes, the analysis of homozygous effects of the mutation can now begin; if not, possible dominant effects are considered.

Production of transgenic periclinal chimeras in pumpkin - a tool for revealing cell fates of L1 meristem.

Genetic engineering is commonly used to improve the agronomic traits of crops. However, genetic transformation in pumpkin remains a challenge. Conducting transformation trials, we accidentally created transgenic L1 periclinal chimeras in pumpkins. Using our modified Agrobacterium-mediated transformation, we generated transgenic L1 periclinal chimeras which have high value in research on development of the meristem. Fluorescence observations of transformed L1 cells enabled us to reveal cell fates. These L1 cells can develop into stomata, epidermal hairs, seed coat, and epidermis of the root, stem, leaf, flower, and fruit. These periclinal chimeras can be propagated vegetatively with minimal risk of transgene flow. This study offers new perspectives on development of the meristem and a promising technique for creating transgenic periclinal chimeras in plants.

Embryonic Stem Cell Gene Targeting and Chimera Production in Mice.

Producing a custom gene mutation in embryonic stem (ES) cells, whether through homologous recombination or CRISPR-Cas gene editing, is the first step along the way to getting the mutation into live mice. However, there are a number of additional steps along the way, each presenting technical challenges. Here, we provide a guide for troubleshooting when the results are not as expected and to distinguish technical problems from possible biological effects of the mutation. From the isolation of clonal lines of targeted ES cells through the production of ES cell chimeras with the targeted ES cell clone, we discuss common technical problems and their most likely causes and solutions. We also provide guidance for situations where the mutation has a phenotype in the form of a dominant effect on ES cells or in chimeras.

First study on repeatable culture of primordial germ cells from various embryonic regions with giant feeder cells in Japanese quail (Coturnix japonica)

Japanese quail (JQ, Coturnix japonica) is a farmed animal with a high economic value and has been used extensively as an avian model for research. Germline chimera production based on cryopreserved primordial germ cells (PGCs) is possible for conservation management of quail breeds as successful isolation has been reported of PGCs from their blood and gonads. However, the repeatable cultivation protocol has not been elucidated yet, which has hindered technological development. The current study characterized cultivation of pregonadal PGCs isolated from embryonic parts; embryonic blood (cPGCs), whole embryonic tissues (tPGCs), parts of tail buds (tbPGCs), and a mixture of blood and tail bud tissues (ctbPGCs). The results showed that the cultivation system required the presence of specific embryonic cells to act as a feeder for JQ-PGCs and that such a system facilitated more successful cultivation, as shown by the percentages of isolation and cultivation in tbPGCs (100%, 100%, respectively), tPGCs (60%, 55%, respectively), and ctbPGCs (60%, 30%, respectively), but not in cPGCs (0%) cultured on a mitomycin-treated JQ feeder cell-line. Once the co-culture system had been established, the PGCs could be propagated for at least 5 months. These PGCs expressed germ cell-specific markers (DAZL and CVH) and could colonize embryonic gonads. Conclusively, the isolation of pregonadal PGCs and their long-term cultivation in vitro requires a unique embryonic cell, giant cell feeder, that is indispensable for the proliferation of PGCs. Characterization of cell signaling sustaining a mutual interaction between the PGCs and the specific feeder cells will elucidate a superior environment for in vitro cultivation, as well as support the minimal transfer of used xenobiotics in chimera production.

Review: Progress in producing chimeric ungulate livestock for agricultural applications.

The progress made in recent years in the derivation and culture of pluripotent stem cells from farm animals opens up the possibility of creating livestock chimeras. Chimeras producing gametes exclusively derived from elite donor stem cells could pass superior genetics on to the next generation and thereby reduce the genetic lag that typically exists between the elite breeding sector and the commercial production sector, especially for industries like beef and sheep where genetics is commonly disseminated through natural service mating. Chimeras carrying germ cells generated from genome-edited or genetically engineered pluripotent stem cells could further disseminate useful genomic alterations such as climate adaptation, animal welfare improvements, the repair of deleterious genetic conditions, and/or the elimination of undesired traits such as disease susceptibility to the next generation. Despite the successful production of chimeras with germ cells generated from pluripotent donor stem cells injected into preimplantation-stage blastocysts in model species, there are no documented cases of this occurring in livestock. Here, we review the literature on the derivation of pluripotent stem cells from ungulates, and progress in the production of chimeric ungulate livestock for agricultural applications, drawing on insights from studies done in model species, and discuss future possibilities of this fast-moving and developing field. Aside from the technical aspects, the consistency of the regulatory approach taken by different jurisdictions towards chimeric ungulate livestock with germ cells generated from pluripotent stem cells and their progeny will be an important determinant of breeding industry uptake and adoption in animal agriculture.

Current Approaches to and the Application of Intracytoplasmic Sperm Injection (ICSI) for Avian Genome Editing.

Poultry are one of the most valuable resources for human society. They are also recognized as a powerful experimental animal for basic research on embryogenesis. Demands for the supply of low-allergen eggs and bioreactors have increased with the development of programmable genome editing technology. The CRISPR/Cas9 system has recently been used to produce transgenic animals and various animals in the agricultural industry and has also been successfully adopted for the modification of chicken and quail genomes. In this review, we describe the successful establishment of genome-edited lines combined with germline chimera production systems mediated by primordial germ cells and by viral infection in poultry. The avian intracytoplasmic sperm injection (ICSI) system that we previously established and recent advances in ICSI for genome editing are also summarized.

Human-animal chimeras. A systematic review of the ethical problems they raise and their possible solutions.

Human-animal chimera research has gradually evolved to the present day, in which large projects related to the attempt to solve pathologies that help us human beings to alleviate diseases. However, it must be considered that many of these advances in science imply an important ethical dilemma in many cases, and even more so if we involve people in said experiments. In the present systematic review we sought to identify these ethical problems related to chimeras, as well as possible solutions to them proposed in the literature, including technical means for the realization of less humanized chimeras. A bibliographic search was carried out in the Pubmed, Embase and Medes databases on January 4 th, 2022. The articles that strictly comply with the objectives selected for the completion of the work will be selected. A total of 21 articles makes up our sample, from which ethical problems related to chimeras, possible solutions and technical means to avoid obtaining too humanized chimeras will be extracted. The issues identified in the articles are problems related to animal welfare, acquisition of human traits from chimeras, medical concerns derived from experimentation such as zoonoses, the origin of pluripotential cells for chimera production, the creation of human gametes by said chimeras, neurological chimerism and the moral status of chimeras. This paper provides solutions for these problems, such as the use of suicide genes in human cells that would be activated if they differentiate into neuronal cells or the use of gene editing through the CRISPR/Cas9 mechanism to incapacitate these cells so that they do not differentiate into neuronal cells. The only question that remains elusive to the proposal of solutions is the one related to the potential moral status of chimeras. It is certainly a complex issue given the variety of proposals on the concept of moral status available in literature. It is therefore necessary to bring these proposals closer to reflection on human-animal chimeras in order to initiate a discussion that can shed light on this issue.

Efficient simultaneous double DNA knock-in in murine embryonic stem cells by CRISPR/Cas9 ribonucleoprotein-mediated circular plasmid targeting for generating gene-manipulated mice

Gene targeting of embryonic stem (ES) cells followed by chimera production has been conventionally used for developing gene-manipulated mice. Although direct knock-in (KI) using murine zygote via CRISPR/Cas9-mediated genome editing has been reported, ES cell targeting still has merits, e.g., high throughput work can be performed in vitro. In this study, we first compared the KI efficiency of mouse ES cells with CRISPR/Cas9 expression vector and ribonucleoprotein (RNP), and confirmed that KI efficiency was significantly increased by using RNP. Using CRISPR/Cas9 RNP and circular plasmid with homologous arms as a targeting vector, knock-in within ES cell clones could be obtained efficiently without drug selection, thus potentially shortening the vector construction or cell culture period. Moreover, by incorporating a drug-resistant cassette into the targeting vectors, double DNA KI can be simultaneously achieved at high efficiency by a single electroporation. This technique will help to facilitate the production of genetically modified mouse models that are fundamental for exploring topics related to human and mammalian biology.

Transplantation and enrichment of busulfan-resistant primordial germ cells into adult testes for efficient production of germline chimeras in songbirds†.

Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production.

Primordial germ cell identification and traceability during the initial development of the Siluriformes fish Pseudopimelodus mangurus.

Primordial germ cells (PGCs) are responsible for generating all germ cells. Therefore, they are essential targets to be used as a tool for the production of germline chimeras. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. A specific antisense RNA probe constituted by a partial coding region from P. mangurus nanos3 mRNA was synthesized for the WISH method. RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus buc and nanos3 3'UTR sequences, germline-specific markers used to describe in vivo migration of PGCs. Nanos3 and buc gene expression was evaluated in tissues for male and female adults and initial development phases and larvae from the first to seventh days post-hatching. The results from the WISH technique indicated the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained from nanos3 probes corresponded topographically to the migratory patterns of the PGCs reported for other fish species. Diffuse signals were observed in all blastomeres until the 16-cell stage, which could be related to the two sequences of the nanos3 3'UTR observed in the P. mangurus unfertilized egg transcriptome. Microinjection was not successful using GFP-Dr-nanos1 3'UTR mRNA and GFP-Pm-buc 3'UTR mRNA and allowed the identification of potential PGCs with less than 2% efficiency only and after hatching using GFP-Pm-nanos3 3'UTR. Nanos3 and buc gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimeras to be applied in the conservation efforts of Neotropical Siluriformes species. This study can contribute to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as transplanting germ cells in fish.

Can cloning and sequencing help to genotype positive Toxoplasma gondii clinical samples? Results and validation using SAG3 as a model.

Genotyping of T. gondii in human cases is relevant to understand the transmission patterns and epidemiology of this parasitosis. However, this genetic characterization can be hampered by the difficulty of isolating the parasite from mild or asymptomatic cases and by the detection efficiency of molecular assays such as the multilocus nested-polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RLFP). To propose an alternative for the genotyping of positive clinical samples of T. gondii with a low amount of the parasite DNA mixed within the host DNA or mixed infections, we carried out this study to validate the sequences of the SAG3 gene of T. gondii obtained after two rounds of amplification cloned into a bacterial model, thereby achieving the separation and identification of more than one genotype of T. gondii. Also, the detection limit of the parasite DNA and the fidelity of the reagents used in the nested PCR-RFLP in artificial clinical samples by sequencing were determined. T. gondii DNA was detected from 6.25ng of DNA and 200 parasites/mL of blood. The fidelity of the AmpliTaq Gold™ polymerase after 65cycles of amplification was 100%. Denaturation of the products obtained after two rounds of nested PCR amplification showed no evidence of chimera or artifact production. The cloning efficiency was 97.5% (39/40 clones), and none of the experiments produced recombinant sequences. Thus, the generation of chimeras with this methodology could be ruled out. Genotyping of clinical samples is important because there is no strain selection bias, as can occur in the bioassay (where more virulent strains can be selected over nonvirulent strains), and therefore, mixed infections can be detected through cloning and sequencing. Furthermore, these two techniques could be useful tools to genotype weak amplicons of any T. gondii gene obtained during nested PCR.

Production and Immunogenicity of a Tag-Free Recombinant Chimera Based on PfMSP-1 and PfMSP-3 Using Alhydrogel and Dipeptide-Based Hydrogels.

A fusion chimeric vaccine comprising multiple protective domains of different blood-stage Plasmodium falciparum antigens is perhaps necessary for widening the protective immune responses and reducing the morbidity caused by the disease. Here we continue to build upon the prior work of developing a recombinant fusion chimera protein, His-tagged PfMSP-Fu24, by producing it as a tag-free recombinant protein. In this study, tag-free recombinant PfMSPFu24 (rFu24) was expressed in Escherichia coli, and the soluble protein was purified using a three-step purification involving ammonium sulphate precipitation followed by 2-step ion exchange chromatography procedures and shown that it was highly immunogenic with the human-compatible adjuvant Alhydrogel. We further investigated two dipeptides, phenylalanine-α, β-dehydrophenylalanine (FΔF) and Leucine-α, β-dehydrophenylalanine (LΔF) based hydrogels as effective delivery platforms for rFu24. These dipeptides self-assembled spontaneously to form a highly stable hydrogel under physiological conditions. rFu24 was efficiently entrapped in both the F∆F and L∆F hydrogels, and the three-dimensional (3D) mesh-like structures of the hydrogels remained intact after the entrapment of the antigen. The two hydrogels significantly stimulated rFu24-specific antibody titers, and the sera from the immunized mice showed an invasion inhibitory activity comparable to that of Alhydrogel. Easily synthesized dipeptide hydrogels can be used as an effective antigen delivery platform to induce immune responses.

New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing.

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

Current Approaches and Applications in Avian Genome Editing.

Advances in genome-editing technologies and sequencing of animal genomes enable researchers to generate genome-edited (GE) livestock as valuable animal models that benefit biological researches and biomedical and agricultural industries. As birds are an important species in biology and agriculture, their genome editing has gained significant interest and is mainly performed by using a primordial germ cell (PGC)-mediated method because pronuclear injection is not practical in the avian species. In this method, PGCs can be isolated, cultured, genetically edited in vitro, and injected into a recipient embryo to produce GE offspring. Recently, a couple of GE quail have been generated by using the newly developed adenovirus-mediated method. Without technically required in vitro procedures of the PGC-mediated method, direct injection of adenovirus into the avian blastoderm in the freshly laid eggs resulted in the production of germ-line chimera and GE offspring. As more approaches are available in avian genome editing, avian research in various fields will progress rapidly. In this review, we describe the development of avian genome editing and scientific and industrial applications of GE avian species.

Totipotency continuity from zygote to early blastomeres: a model under revision.

The mammalian zygote is a totipotent cell that generates all the cells of a new organism through embryonic development. However, if one asks about the totipotency of blastomeres after one or two zygotic divisions, opinions differ. As it is impossible to determine the individual developmental potency of early blastomeres in an intact embryo, experiments of blastomere isolation were conducted in various species, showing that two-cell blastomeres could give rise to a new organism when sister cells were separated. A mainstream interpretation was that each of the sister mammalian blastomeres was equally totipotent. However, reevaluation of those experiments raised some doubts about the real prevalence of cases in which this interpretation could truly be validated. We compiled experiments that tested the individual developmental potency of early mammalian blastomeres in a cell-autonomous way (i.e. excluding nuclear transfer and chimera production). We then confronted the developmental abilities with reported molecular differences between sister blastomeres. The reevaluated observations were at odds with the mainstream view: A viable two-cell embryo can already include one non-totipotent blastomere. We were, thus, led to propose a revised model for totipotency continuity based on the construction of the zygote as a mosaic, which accounts for differential inheritance of totipotency-relevant components between sister blastomeres. This takes place with no preordained mechanisms that would ensure a reproducible partition. This model, which is compatible with the body of data on regulative properties of mammalian early embryos, aims at tempering the rigid interpretation that discounted maternal constraints on totipotency.

The Pluripotency Continuum and Interspecies Chimeras.

Pluripotency refers to the capacity of single cells to form derivatives of the three germ layers-ectoderm, mesoderm, and endoderm. Pluripotency can be captured in vitro as a spectrum of pluripotent stem cell states stabilized in specialized laboratory conditions. The recent discovery that pluripotent stem cells can colonize the embryos of distantly related animal organisms could, with further refinement, enable the generation of chimeric embryos composed of cells of human and animal origin. If achievable, the production of human-animal chimeras will open up new opportunities for regenerative medicine, facilitating human disease modeling and human organ generation inside large animals. However, the generation of human-animal interspecies chimeras is anticipated to require human chimera-competent pluripotent stem cells. Thus, it remains imperative to examine the pluripotency continuum more closely in light of advances that will facilitate the production of human-animal chimeras. This piece will review the current understanding of the pluripotency continuum and interspecies chimeras. © 2019 by John Wiley & Sons, Inc.

Development of blastocyst complementation technology without contributions to gametes and the brain.

In Japan, it is possible to generate chimeric animals from specified embryos by combining animal blastocysts with human pluripotent stem (PS) cells (animal-human PS chimera). However, the production of animal-human PS chimeras has been restricted because of ethical concerns, such as the development of human-like intelligence and formation of humanized gametes in the animals, owing to the contributions of human PS cells to the brain and reproductive organs. To solve these problems, we established a novel blastocyst complementation technology that does not contribute to the gametes or the brain. First, we established GFP-expressing mouse embryonic stem cells (G-mESCs) in which the Prdm14 and Otx2 genes were knocked out and generated chimeric mice by injecting them into PDX-1-deficient blastocysts. The results showed that the G-mESCs did not contribute to the formation of gametes and the brain. Therefore, in the PDX-1-deficient mice complemented by G-mESCs without the Prdm14 and Otx2 genes, the germline was not transmitted to the next generations. This approach could address concerns regarding the development of both human gametes and a human-like brain upon mouse blastocyst complementation using human stem cells.

Production of Germ-Line Chimeras in Zebrafish.

The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.

Production of Transgenic Chickens Using Cultured Primordial Germ Cells and Gonocytes.

The unique characteristics of the avian embryo, with its large opaque yolk, have necessitated the development of different approaches to transgenesis from those that have been successful in mammalian species. Genetic modification of birds was greatly advanced by the ability to grow long-term cultures of primordial germ cells (PGCs). These cells are obtained from embryos, established in culture, and can be propagated without losing the ability to contribute to the germline when reintroduced into a host animal. PGCs can be genetically modified in culture using traditional transfection and selection techniques, including gene targeting and site-specific nuclease approaches. Here, we describe our methods for deriving cell lines, long-term culture, genetic modification, production of germline chimeras and obtaining fully transgenic birds with the desired genetic modifications.