An Electrochemical Protocol for CRISPR-Mediated Gene-Editing of Sheep Embryonic Fibroblast Cells.

Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability has remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found 2 pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3% dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in >98% transfection efficiency and >97% cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52% indels rate using single gRNAs as well as a highly efficient target deletion using 2 gRNAs. In conclusion, we have developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3% DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.

Clinical potential of human-induced pluripotent stem cells : Perspectives of induced pluripotent stem cells.

The recent establishment of induced pluripotent stem (iPS) cells promises the development of autologous cell therapies for degenerative diseases, without the ethical concerns associated with human embryonic stem (ES) cells. Initially, iPS cells were generated by retroviral transduction of somatic cells with core reprogramming genes. To avoid potential genotoxic effects associated with retroviral transfection, more recently, alternative non-viral gene transfer approaches were developed. Before a potential clinical application of iPS cell-derived therapies can be planned, it must be ensured that the reprogramming to pluripotency is not associated with genome mutagenesis or epigenetic aberrations. This may include direct effects of the reprogramming method or "off-target" effects associated with the reprogramming or the culture conditions. Thus, a rigorous safety testing of iPS or iPS-derived cells is imperative, including long-term studies in model animals. This will include not only rodents but also larger mammalian model species to allow for assessing long-term stability of the transplanted cells, functional integration into the host tissue, and freedom from undifferentiated iPS cells. Determination of the necessary cell dose is also critical; it is assumed that a minimum of 1 billion transplantable cells is required to achieve a therapeutic effect. This will request medium to long-term in vitro cultivation and dozens of cell divisions, bearing the risk of accumulating replication errors. Here, we review the clinical potential of human iPS cells and evaluate which are the most suitable approaches to overcome or minimize risks associated with the application of iPS cell-derived cell therapies.

Identification and re-addressing of a transcriptionally permissive locus in the porcine genome.

Recently, we established the Sleeping Beauty transposon system for germ line competent transgenesis in the pig. Here, we extend this approach to re-target a transposon-tagged locus for a site-specific gene knock-in, and generated a syngeneic cohort of piglets carrying either the original transposon or the re-targeted event. A Cre-loxP-mediated cassette exchange of the tagging transposon with a different reporter gene was performed, followed by flow cytometric sorting and somatic cell nuclear transfer of recombined cells. In parallel, the original cells were employed in somatic cell nuclear transfer to generate clone siblings, thereby resulting in a clone cohort of piglets carrying different reporter transposons at an identical chromosomal location. Importantly, this strategy supersedes the need for an antibiotic selection marker. This approach expands the arsenal of genome engineering technologies in domestic animals, and will facilitate the development of large animal models for human diseases. Potentially, the syngeneic cohort of pigs will be instrumental for vital tracking of transplanted cells in pre-clinical assessments of novel cell therapies.

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals.

Transgenic farm animals are attractive alternative mammalian models to rodents for the study of developmental, genetic, reproductive and disease-related biological questions, as well for the production of recombinant proteins, or the assessment of xenotransplants for human patients. Until recently, the ability to generate transgenic farm animals relied on methods of passive transgenesis. In recent years, significant improvements have been made to introduce and apply active techniques of transgenesis and genetic engineering in these species. These new approaches dramatically enhance the ease and speed with which livestock species can be genetically modified, and allow to performing precise genetic modifications. This paper provides a synopsis of enzyme-mediated genetic engineering in livestock species covering the early attempts employing naturally occurring DNA-modifying proteins to recent approaches working with tailored enzymatic systems.

Non-viral reprogramming of fibroblasts into induced pluripotent stem cells by Sleeping Beauty and piggyBac transposons.

The generation of induced pluripotent stem (iPS) cells represents a promising approach for innovative cell therapies. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity. Transposition of reprogramming cassettes represents a recent alternative to viral approaches. Since binary transposons can be produced as common plasmids they provide a safe and cost-efficient alternative to viral delivery methods. Here, we compared the efficiency of two different transposon systems, Sleeping Beauty (SB) and piggyBac (PB), for the generation of murine iPS. Murine fibroblasts derived from an inbred BL/6 mouse line carrying a pluripotency reporter, Oct4-EGFP, and fibroblasts derived from outbred NMRI mice were employed for reprogramming. Both transposon systems resulted in the successful isolation of murine iPS cell lines. The reduction of the core reprogramming factors to omit the proto-oncogene c-Myc was compatible with iPS cell line derivation, albeit with reduced reprogramming efficiencies. The transposon-derived iPS cells featured typical hallmarks of pluripotency, including teratoma growth in immunodeficient mice. Thus SB and PB transposons represent a promising non-viral approach for iPS cell derivation.

Reprotoxicity of gold, silver, and gold-silver alloy nanoparticles on mammalian gametes.

Metal and alloy nanoparticles are increasingly developed for biomedical applications, while a firm understanding of their biocompatibility is still missing. Various properties have been reported to influence the toxic potential of nanoparticles. This study aimed to assess the impact of nanoparticle size, surface ligands and chemical composition of gold, silver or gold-silver alloy nanoparticles on mammalian gametes. An in vitro assay for porcine gametes was developed, since these are delicate primary cells, for which well-established culture systems exist and functional parameters are defined. During coincubation with oocytes for 46 h neither any of the tested gold nanoparticles nor the gold-silver alloy particles with a silver molar fraction of up to 50% showed any impact on oocyte maturation. Alloy nanoparticles with 80% silver molar fraction and pure silver nanoparticles inhibited cumulus-oocyte maturation. Confocal microscopy revealed a selective uptake of gold nanoparticles by oocytes, while silver and alloy particles mainly accumulated in the cumulus cell layer surrounding the oocyte. Interestingly sperm vitality parameters (motility, membrane integrity and morphology) were not affected by any of the tested nanoparticles. Only sporadic association of nanoparticles with the sperm plasma membrane was found by transmission electron microscopy. In conclusion, mammalian oocytes were sensitive to silver containing nanoparticles. Likely, the delicate process of completing meiosis in maternal gametes features high vulnerability towards nanomaterial derived toxicity. The results imply that released Ag(+)-ions are responsible for the observed toxicity, but the compounding into an alloy seemed to alleviate the toxic effects to a certain extent.

Biomedical applications of ovarian transvaginal ultrasonography in cattle.

Ovarian transvaginal ultrasonography (OTU) has been used world-wide for commercial ovum pick-up programs for in vitro embryo production in elite herds, providing an excellent model for the elucidation of factors controlling bovine oocyte developmental competence. Noninvasive sampling and treatment of ovarian structures is easily accomplished with bovine OTU techniques providing a promising system for in vivo delivery of transgenes directly into the ovary. The current review summarizes existing bovine OTU models and provides prospective applications of bovine OTU to undertake research in reproductive topics of biomedical relevance, with special emphasis on the development of in vivo gene transfer strategies.

Induced Pluripotent Stem Cells in the Era of Precise Genome Editing.

Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems.

CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII.

On-target integration of large cassettes via homology-directed repair (HDR) has several applications. However, the HDR-mediated targeted knock-in suffered from low efficiency. In this study, we made several large plasmids (12.1-13.4 kb) which included the CRISPR/Cas9 system along with a puromycin transgene as part of the large DNA donor (5.3-7.1 kb insertion cassettes) and used them to evaluate their targeted integration efficiency into a transgenic murine embryonic fibroblast (MEF) cell line carrying a single copy of a Venus transgene. We established a detection assay by which HDR events could be discriminated from the error-prone non-homologous end-joining (NHEJ) events. Improving the plasmid quality could considerably leverage the cell toxicity impediment of large plasmids. The use of the TILD (targeted integration with linearized dsDNA) cassettes did not improve the HDR rate compared to the circular plasmids. However, the direct inclusion of nocodazole into the electroporation solution significantly improved the HDR rate. Also, simultaneous delivery of RNase HII and the donor plasmids into the electroporated cells considerably improved the HDR events. In conclusion, the results of this study showed that using cell synchronization reagents in the electroporation medium can efficiently induce HDR rate in the mammalian genome.

Identification of chicken LOC420478 as Bucky ball equivalent and potential germ plasm organizer in birds.

Bucky ball was identified as germ plasm organizer in zebrafish and has proven crucial for Balbiani body condensation. A synteny comparison identified an uncharacterized gene locus in the chicken genome as predicted avian counterpart. Here, we present experimental evidence that this gene locus indeed encodes a 'Bucky ball' equivalent in matured oocytes and early embryos of chicken. Heterologous expression of Bucky ball fusion proteins both from zebrafish and chicken with a fluorescent reporter revealed unique patterns indicative for liquid-liquid phase separation of intrinsically disordered proteins. Immuno-labeling detected Bucky ball from oocytes to blastoderms with diffuse distribution in matured oocytes, aggregation in first cleavage furrows, and co-localization to the chicken vasa homolog (CVH). Later, Bucky ball translocated to the cytoplasm of first established cells, and showed nuclear translocation during the major zygotic activation together with CVH. Remarkably, during the phase of area pellucida formation, Bucky ball translocated back into the cytoplasm at stage EGK VI, whereas CVH remained within the nuclei. The condensation of Bucky ball and co-localization with CVH in cleavage furrows and nuclei of the centrally located cells strongly suggests chicken Bucky ball as a germ plasm organizer in birds, and indicate a special importance of the major zygotic activation for germline specification.

Generation of Murine Induced Pluripotent Stem Cells through Transposon-Mediated Reprogramming.

The seminal discovery of induced pluripotent stem (iPS) cells through ectopic expression of a cocktail of gene factors (OCT4, SOX2, KLF4, and c-MYC) by the group of Yamanaka was a major breakthrough, gained widespread acclaim and garnered much attention in the field of stem cell science. The iPS cells possess most of the characteristics and advantages of embryonic stem (ES) cells without the association of ethical stigma for their derivation. In addition, these cells can give rise to any cell type of the body and thus have tremendous potential for many downstream applications in research and regenerative medicine. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of oncogenicity and insertional mutagenesis. Nonviral methods for generation of iPS cells through somatic cell reprogramming are powerful tools for establishing in vitro disease models, development of new protocols for treatment of different diseases, and creating transgenic mice models. Here, we present a detailed protocol for the generation of transposon-mediated iPS cells from mouse embryonic fibroblasts (MEFs) and give a short overview of the characterization of the generated iPS cell lines.

Applications of Genome Editing Tools in Stem Cells Towards Regenerative Medicine: An Update.

Precise and site-specific genome editing through application of emerging and modern gene engineering techniques, namely zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/ Cas9) have swiftly progressed the application and use of the stem cell technology in the sphere of in-vitro disease modelling and regenerative medicine. Genome editing tools facilitate the manipulation of genes in various types of cells with target-specific nucleases. These tools aid in elucidating the genetics and etiology behind different diseases and have immense promise as novel therapeutics for correcting the genetic mutations, making alterations, and curing diseases permanently, which are not responding and resistant to traditional therapies. These genome engineering tools have evolved in the field of biomedical research and have also been shown to have a significant improvement in clinical trials. However, their widespread use in the research revealed potential safety issues, which need to be addressed before implementing such techniques for clinical purposes. Significant and valiant attempts need to be made in order to surpass those hurdles. The current review outlines the advancements of several genome engineering tools and describes suitable strategies for their application towards regenerative medicine.

Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR.

Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5'-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs.

Species-specific telomere length differences between blastocyst cell compartments and ectopic telomere extension in early bovine embryos by human telomerase reverse transcriptase.

The enzyme telomerase is active in germ cells and is critically involved in maintenance of telomere length in successive generations. In preimplantation mammalian embryos, telomerase activity is present from the morula stage onward and is associated with an increase in telomere length in blastocysts. Herein, we show that telomere length regulation in murine and bovine blastocysts differed between trophectodermal and inner cell mass cells in a species-specific manner. Ectopic expression of human telomerase reverse transcriptase (TERT) in bovine embryos increased telomerase activity and in turn increased telomere length. Transient expression of human TERT could be targeted to the 4-cell to morula stages and to the morula to blastocyst stages using unmodified and cytosine-methylated expression plasmids, respectively. Introduction of human TERT constructs in bovine embryos resulted in functional telomerase expression and effective telomere elongation, allowing us to study the effects on embryonic development. Ultimately, these studies may lead to a large-animal model for telomere regulation and aging.

In vivo oocyte developmental competence is reduced in lean but not in obese superovulated dairy cows after intraovarian administration of IGF1.

The present study investigated the role of IGF1 in lactating lean and non-lactating obese dairy cows by injecting 1 µg IGF1 into the ovaries prior to superovulation. This amount of IGF1 has been linked with pregnancy loss in women with the polycystic ovary syndrome (PCOS) and was associated with impaired bovine oocyte competence in vitro. Transcript abundance and protein expression of selected genes involved in apoptosis, glucose metabolism, and the IGF system were analyzed. Plasma concentrations of IGF1 and leptin, and IGF1 in uterine luminal fluid (ULF), were also measured. IGF1 treatment decreased embryo viability in lean cows to the levels observed in obese cows. Obese cows were not affected by IGF1 treatment and showed elevated levels of IGF1 (in both plasma and ULF) and leptin. Blastocysts from lean cows treated with IGF1 showed a higher abundance of SLC2A1 and IGFBP3 transcripts. IGF1 treatment reduced protein expression of tumor protein 53 in blastocysts of lean cows, whereas the opposite was observed in obese cows. IGF1 in plasma and ULF was correlated only in the control groups. Blastocyst transcript abundance of IGF1 receptor and IGFBP3 correlated positively with IGF1 concentrations in both plasma and ULF in lean cows. The detrimental microenvironment created by IGF1 injection in lean cows and the lack of effect in obese cows resemble to a certain extent the situation observed in PCOS patients, where IGF1 bioavailability is increased in normal-weight women but reduced in obese women, suggesting that this bovine model could be useful for studying IGF1 involvement in PCOS.

Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens.

The chicken (Gallus gallus) is one of the most common and widespread domestic species, with an estimated total population of 25 billion birds worldwide. The vast majority of chickens in agriculture originate from hybrid breeding programs and is concentrated on few commercially used high performance lines, whereas numerous local and indigenous breeds are at risk to become extinct. To preserve the genomic resources of rare and endangered chicken breeds innovative methods are necessary. Here, we established a solid workflow for the derivation and biobanking of chicken primordial germ cells (PGCs) from blue layer hybrids. To achieve this, embryos of a cross of heterozygous blue egg layers were sampled to obtain blood derived and gonadal male as well as female PGCs of different genotypes (homozygous, heterozygous and nullizygous blue-allele bearing). The total efficiency of established PGC lines was 45% (47/104) within an average of 49 days until they reached sufficient numbers of cells for cryopreservation. The stem-cell character of the cultivated PGCs was confirmed by SSEA-1 immunostaining, and RT-PCR amplification of the pluripotency- and PGC-specific genes cPOUV, cNANOG, cDAZL and CVH. The Sleeping Beauty transposon system allowed to generate a stable integration of a Venus fluorophore reporter into the chicken genome. Finally, we demonstrated that, after re-transfer into chicken embryos, Venus-positive PGCs migrated and colonized the forming gonads. Semen samples of 13 raised cell chimeric roosters were analyzed by flow cytometry for the efficiency of germline colonization by the transferred PGCs carrying the Venus reporter and their proper differentiation into vital spermatids. Thus, we provide a proof-of-concept study for the potential use of PGCs for the cryobanking of rare breeds or rare alleles.

Perspectives of pluripotent stem cells in livestock.

The recent progress in derivation of pluripotent stem cells (PSCs) from farm animals opens new approaches not only for reproduction, genetic engineering, treatment and conservation of these species, but also for screening novel drugs for their efficacy and toxicity, and modelling of human diseases. Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages, or lost their cellular potency; indicating that the protocols which allowed the derivation of murine or human embryonic stem (ES) cells were not sufficient to support the maintenance of ES cells from farm animals. This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support naïve pluripotency in ES cells from livestock species. However, the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging, and requires further refinements. Here, we review the current achievements in the derivation of PSCs from farm animals, and discuss the potential application areas.

Generation and Breeding of EGFP-Transgenic Marmoset Monkeys: Cell Chimerism and Implications for Disease Modeling.

Genetic modification of non-human primates (NHP) paves the way for realistic disease models. The common marmoset is a NHP species increasingly used in biomedical research. Despite the invention of RNA-guided nucleases, one strategy for protein overexpression in NHP is still lentiviral transduction. We generated three male and one female enhanced green fluorescent protein (EGFP)-transgenic founder marmosets via lentiviral transduction of natural preimplantation embryos. All founders accomplished germline transmission of the transgene by natural mating, yielding 20 transgenic offspring together (in total, 45 pups; 44% transgenic). This demonstrates that the transgenic gametes are capable of natural fertilization even when in competition with wildtype gametes. Importantly, 90% of the transgenic offspring showed transgene silencing, which is in sharp contrast to rodents, where the identical transgene facilitated robust EGFP expression. Furthermore, we consistently discovered somatic, but so far, no germ cell chimerism in mixed wildtype/transgenic litters. Somatic cell chimerism resulted in false-positive genotyping of the respective wildtype littermates. For the discrimination of transgenic from transgene-chimeric animals by polymerase chain reaction on skin samples, a chimeric cell depletion protocol was established. In summary, it is possible to establish a cohort of genetically modified marmosets by natural mating, but specific requirements including careful promoter selection are essential.

Development of a transposon-based technology for transfection of day 0 chicken embryos.

Although the chicken embryo has been a classical model for developmental studies, the lack of straightforward technologies for chicken transgenesis limited the usefulness of this animal model. Here, we assessed electroporation and lipofection approaches for in ovo transfection of Sleeping Beauty transposon system in stage X-XII chicken embryos. Electroporation of chicken embryos could transfect the trophectodermal cells. Then, a mixture of transposon lipoplexes and high concentrated carboxymethylcellulose (HCC) solution was injected into the subgerminal cavity of day 0 embryos. The lipoplex-HCC mixture substantially increased the number of trophectodermal cells expressing the reporter. Importantly, the fluorescent reporter was detected in cells inside of the embryos as well as circulation cells in the bloodstream during days 3-4 of incubation. This study provided evidence for direct in ovo transfection of early chicken embryos, though the long-term outcome of this approach warrants further studies.

A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells.

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.