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Health monitoring of laboratory rodents not only improves animal health but also enhances the validity of animal experiments. In particular, infections of laboratory animals with murine parvoviruses influence biomedical research data. Despite strict barrier housing, prevalence remains high in animal facilities, leading to increased risk of parvovirus introduction after the import of contaminated mice. Unfortunately, hygienic rederivation can be challenging, since gametes often contain residual virus material. Consequently, the process has to be closely monitored with highly sensitive diagnostic methods to verify parvovirus decontamination of the rederived progeny. However, diagnostic sensitivity of traditional methods is often low and requires testing of large animal cohorts. Therefore, we aimed to develop a powerful quantitative real-time polymerase chain reaction (qPCR) assay for the fast and reliable detection of murine parvoviruses in different sample materials. We validated the assay within an infection experiment and systematically analysed various animal-derived and environmental sample materials. We further developed a strategic risk assessment procedure for parvovirus monitoring after embryo transfer. Our novel qPCR assay reliably detected parvovirus DNA in a broad variety of sample materials, with environmental samples dominating in the acute phase of infection, whereas animal-derived samples were more suitable to detect low virus loads in the chronic phase. Here, the assay served as a highly sensitive screening method for parvovirus contamination in mouse colonies, requiring significantly lower sample sizes than traditional methods like conventional PCR and serology. Thus, the use of our novel qPCR assay substantially improves parvovirus diagnostics, enhancing research validity according to the 6Rs.

For a wide range of biomedical approaches, an accurate estimate of the age of embryos or pups is important. Overnight mating is the method that is mostly used to establish timed pregnancies. The oestrus cycle in mice repeats every four to five days. So, not all females will get pregnant because they are not in oestrus. Therefore, the aim of this study was to analyse whether polygamous mating could increase the rate of timed pregnancies per breeding cage and female. We compared overnight timed mating regimes with up to four females per male, using C57BL/6 and BALB/c mice as well as F1 hybrids of these two strains. The number of vaginal plugs, number of females that gave birth and weaned litter (including size and weaning weight) were recorded. Our results showed that the plug and pregnancy rate decreased, but the productivity per breeding cage increased for polygamous mating regimes. The proportion of females with vaginal plugs and females that gave birth was significantly higher in monogamous mating. The proportion of plugged females that gave birth, as well as litter size and weaning weight, were not influenced by the mating regime. After analysing 513 breeding cages with a total of 1090 females, we found that polygamous mating with up to three females per male can increase the number of timed pregnancies. However, in the mating regime with more than three females, the rate of timed pregnancy as well as number of pups per female declined.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study, we focus on the properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing (scRNAseq) of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study we focus on the profibrotic properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNA seq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrated that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.

Various laboratory animals have been used for understanding human diseases. Most popular laboratory animals are rodents, such as mouse and rat. Reproductive technologies have been developed for efficient production and stable long-term preservation as genetic resources of these strains. In this review, the current status of the various reproductive technologies has been discussed.

The laboratory mouse is the most used animal model in biomedical research. Several artificial reproductive techniques, such as revitalization of cryopreserved strains, rederivation after hygienic contaminations and the production of transgenic mouse models, require the transfer of preimplantation embryos to surrogate mothers. Pseudopregnancy is essential in recipient females and is induced by mating with sterile males. Commonly, surgically vasectomized males are used for this purpose. As an alternative, genetically modified mouse strains have been identified, in which homozygous infertile males are sexually active. Here, we investigated the suitability of genetically infertile Gapdhstm1Dao males under routine laboratory conditions with respect to plug rates, pregnancy rates and frequency of born offspring after embryo transfer. Our results showed no significant differences for these aspects between Gapdhstm1Dao and vasectomized CD2F1 males. In addition, we evaluated the efforts to obtain a defined number of sterile males either by breeding of sterile mutants or surgical vasectomy, and addressed the impact of both options on animal welfare. In conclusion, infertile males of the Gapdhstm1Dao line are a reliable alternative to vasectomized males for the induction of pseudopregnancy, and can contribute to the refinement of the procedure by avoiding surgical interventions.

Embryo transfer of pre-implantation embryos to surrogate dams is a key technique for the hygienic sanitation of strains, cryopreservation, in vitro fertilization, genetic modification and engineering. However, the effects of several parameters, such as the number of transferred embryos, on the success of embryo transfer are not well studied. In this retrospective study, we reanalysed 1320 embryo transfers of two-cell embryos originating from genetically altered donors, which were performed under routine conditions in our facility over a period of 5 years. Of them, 453 embryo transfers were done with freshly collected embryos and 867 transfers were performed with cryopreserved embryos. Despite the fact that the genetic background of the embryo donors was quite heterogeneous, we found that the transfer of ≥ 21 embryos reduced the success of embryo transfers for freshly collected embryos in correlation with the number of pregnancies and born pups, whereas this was not the case for transfer in the cryopreservation group. Most pregnancies were achieved after embryo transfer of 10–20 freshly collected embryos (90.4%), which dropped to 37.5% if more embryos were transferred. The highest pregnancy rates in the cryopreservation group were achieved if 15–17 embryos were transferred (62.9%). Despite the fact that the precise substrains were only rarely defined, we confirmed that beside the number of transferred embryos, the genetic background of the donors had an influence on the success of embryo transfer. Significantly more embryos in a C57BL/6 background developed to term than embryos on a BALB/c background.

The incidence of obesity is on the rise in most western countries and represents major risks to health. Obesity causes complex metabolic dysfunctions and can be associated with a large number of secondary diseases. To investigate causal mechanisms of obesity and develop better options for treatment, researchers study the condition in animal models. In addition to genetically engineered animal models, diet-induced obesity is often used because it occurs similarly in animals as it does in humans. For several types of investigations that use obesity models, investigators must carry out surgical interventions and they frequently encounter severe perioperative complications induced by anesthesia. In an example of this problem, we observed 100% mortality in obese BALB/c mice after ovariectomy, despite no obvious surgical complications. We supposed that a failure to recover from surgery was the primary cause of this increased mortality. Therefore, to support their recovery from surgery we administered atropine to obese mice in order to facilitate blood circulation, and we also increased the oxygen content of the ambient air. With this specific support before and after surgery, we increased the survival rate of obese ovariectomized mice up to 83%. These results confirm the assumption that obesity is a risk factor for the recovery of obese animal models after ovariectomy, and they highlight the need to provide additional interventions for such experimental animals.

Induced pluripotent stem cells (iPSCs) are a seminal breakthrough in stem cell research and are promising tools for advanced regenerative therapies in humans and reproductive biotechnology in farm animals. iPSCs are particularly valuable in species in which authentic embryonic stem cell (ESC) lines are yet not available. Here, we describe a nonviral method for the derivation of bovine iPSCs employing Sleeping Beauty (SB) and piggyBac (PB) transposon systems encoding different combinations of reprogramming factors, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter. One bovine iPSC line (biPS-1) generated by a PB vector containing six reprogramming genes was analyzed in detail, including morphology, alkaline phosphatase expression, and typical hallmarks of pluripotency, such as expression of pluripotency markers and formation of mature teratomas in immunodeficient mice. Moreover, the biPS-1 line allowed a second round of SB transposon-mediated gene transfer. These results are promising for derivation of germ line–competent bovine iPSCs and will facilitate genetic modification of the bovine genome.

We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNA encoding the SB100X hyperactive transposase, together with circular plasmid DNA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼3 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies to lentiviral approaches without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.

We describe the expression of recombinant fluorescent proteins in the milk of two lines of transgenic pigs generated by Sleeping Beauty transposon-mediated genetic engineering. The Sleeping Beauty transposon consisted of an ubiquitously active CAGGS promoter driving a fluorophore cDNA, encoding either Venus or mCherry. Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found. Unexpectedly, milk samples from lactating sows contained high levels of bioactive Venus or mCherry fluorophores. A detailed analysis suggested that exfoliated cells of the mammary epithelium carried the recombinant proteins passively into the milk. This is the first description of reporter fluorophore expression in the milk of livestock, and the findings may contribute to the development of an alternative concept for the production of bioactive recombinant proteins in the udder.

Genetically modified cattle are important for developing new biomedical models and for an improved understanding of the pathophysiology of zoonotic diseases. However, genome editing and genetic engineering based on somatic cell nuclear transfer suffer from a low overall efficiency. Here, we established a highly efficient one-step multiplex gene transfer system into the bovine genome.

Transgenesis in the mouse is an essential tool for the understanding of gene function and genome organization. Here, we describe a simplified microinjection protocol for efficient germline transgenesis and sustained transgene expression in the mouse model employing binary Sleeping Beauty transposon constructs of different topology. The protocol is based on co‐injection of supercoiled plasmids or minicircles, encoding the Sleeping Beauty transposase and a transposon construct, into the cytoplasm of murine zygotes. Importantly, this simplified injection avoids the mechanical penetration of the vulnerable pronuclear membrane, resulting in higher survival rates of treated embryos and a more rapid pace of injections. Upon translation of the transposase, transposase‐catalyzed transposition into the genome results in stable transgenic animals carrying monomeric transgenes. In summary, cytoplasmic injection of binary transposon constructs is a feasible, plasmid‐based, and simplified microinjection method to generate genetically modified mice. The modular design of the components allows the multiplexing of different transposons, and the generation of multi‐transposon transgenic mice in a single step. Fil: Garrels, Wiebke. Friedrich Loeffler Institut; Alemania Fil: Talluri, Thirumala R.. Friedrich Loeffler Institut; Alemania Fil: Ziegler, Maren. Friedrich Loeffler Institut; Alemania Fil: Most, Ilka. Friedrich Loeffler Institut; Alemania Fil: Forcato, Diego Oscar. Friedrich Loeffler Institut; Alemania. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schmeer, Marco. PlasmidFactory; Alemania Fil: Schleef, Martin. PlasmidFactory; Alemania Fil: Ivics, Zoltán. Paul Ehrlich Institute; Alemania Fil: Kues, Wilfried A.. Friedrich Loeffler Institut; Alemania

Recently, we described a simplified injection method for producing transgenic pigs using a non-autonomous Sleeping Beauty transposon system. The founder animals showed ubiquitous expression of the Venus fluorophore in almost all cell types. To assess, whether expression of the reporter fluorophore affects animal welfare or fecundity, we analyzed reproductive parameters of two founder boars, germ line transmission, and organ and cell specific transgene expression in animals of the F1 and F2 generation. Molecular analysis of ejaculated sperm cells suggested three monomeric integrations of the Venus transposon in both founders. To test germ line transmission of the three monomeric transposon integrations, wild-type sows were artificially inseminated. The offspring were nursed to sexual maturity and hemizygous lines were established. A clear segregation of the monomeric transposons following the Mendelian rules was observed in the F1 and F2 offspring. Apparently, almost all somatic cells, as well as oocytes and spermatozoa, expressed the Venus fluorophore at cell-type specific levels. No detrimental effects of Venus expression on animal health or fecundity were found. Importantly, all hemizygous lines expressed the fluorophore in comparable levels, and no case of transgene silencing or variegated expression was found after germ line transmission, suggesting that the insertions occurred at transcriptionally permissive loci. The results show that Sleeping Beauty transposase-catalyzed transposition is a promising approach for stable genetic modification of the pig genome.

Precision genetic engineering based on stable chromosomal insertion of exogenous DNA in the genomes of large mammals is immensely important for the development of improved biomedical models, pharmaceutical research and an accelerated breeding progress. Precision genetic engineering requires (i) a known locus of genomic integration, (ii) a defined status of foreign DNA, (iii) that transgene expression is unaffected by neighbouring chromosomal sequences, (iv) endogenous genes are not mutated and (v) no unwanted DNA sequences are present. Recently, advanced molecular techniques exploiting exogenous enzymes have opened the possibilities for more sophisticated genetic engineering. Here, we critically review current developments of enzyme-catalysed approaches for targeted transgenesis in large mammals.

Here we describe a non-invasive method for rapid and highly reproducible genotyping of transgenic mammals with ubiquitous expression of fluorophore reporters. Hair samples from transgenic mice and pigs with systemic expression of the fluorophore reporter Venus were analyzed with a fluorescence microscope in few minutes. The hair samples can be preserved for long-term storage at ambient temperature conditions. This non-invasive method is useful for genotyping of transgenic large animals and contributes to animal welfare by reducing stress and discomfort of the animals during sample collection.

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.

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.

Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%). Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A) did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag+-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos.

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the piggyBac (PB) and sleeping beauty (SB) transposon systems were assessed for stable gene transfer into the cattle genome. Bovine fibroblasts were transfected either with a helper-independent PB system or a binary SB system. Both transposons were highly active in bovine cells increasing the efficiency of DNA integration up to 88 times over basal nonfacilitated integrations in a colony formation assay. SB transposase catalyzed multiplex transgene integrations in fibroblast cells transfected with the helper vector and two donor vectors carrying different transgenes (fluorophore and neomycin resistance). Stably transfected fibroblasts were used for SCNT and on in vitro embryo culture, morphologically normal blastocysts that expressed the fluorophore were obtained with both transposon systems. The data indicate that transpositionis a feasible approach for genetic engineering in the cattle genome. Fil: Alessio, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Fili, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Garrels, Wiebke. Institut für Nutztiergenetik; Alemania. Gottfried Wilhelm Leibniz Universität Hannover; Alemania Fil: Forcato, Diego Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Olmos Nicotra, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Liaudat, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Bevacqua, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina Fil: Savy, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina Fil: Hiriart, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina Fil: Talluri, Thirumala R.. Institut für Nutztiergenetik; Alemania Fil: Owens, Jesse B.. University of Hawaii at Manoa; Estados Unidos Fil: Ivics, Zoltán. Paul-Ehrlich-Institute; Alemania Fil: Salamone, Daniel Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina Fil: Moisyadi, Stefan. University of Hawaii at Manoa; Estados Unidos Fil: Kues, Wilfried A.. Institut für Nutztiergenetik; Alemania Fil: Bosch, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina

Active transposon-mediated transgenesis is an emerging tool for basic and applied research in livestock. We have demonstrated the effectiveness of a helper-independent piggyBac transposon (pGENIE-3) for gene transfer into the genome of bovine cells (Alessio et al. 2014 Reprod. Domest. Anim. 49, 8). Here, we extend our previous research by examining the suitability of a Sleeping Beauty (SB) transposon-based methodology to deliver transgenes into the genome of bovine fetal fibroblasts (BFF), and the ability of these cells to support in vitro embryo development upon somatic cell nuclear transfer (SCNT). In a first experiment, BFF were chemically cotransfected (JetPRIME®, Polyplus-transfection, Illkirch, France) with a helper plasmid (pCMV-SB100X), which carries an expression cassette for the SB transposase, and the donor vector (pT2/Venus/RMCE) harboring an expression cassette for a fluorescent protein (Venus) flanked by the SB inverted terminal repeats (ITR). Three different ratios of helper and donor plasmids were studied: 1 : 2, 1 : 1 and 2 : 1. After 15 days of culture, the number of fluorescent colonies was counted on an inverted microscope. When vectors were used at ratios of 1 : 1 and 2 : 1, a 78-fold and 88-fold increase (P ≤ 0.05) in the number of fluorescent colonies compared with that in the no-transposase control were calculated. In a second experiment, BFF were chemically cotransfected with the helper vector pCMV-SB100X, and 2 donor transposons: pT2/Venus/RMCE and pT2/SV40-Neo. The former harbors a neo resistance cassette framed by SB ITRs. Different ratios of helper:donors (1 : 1 : 1, 2 : 1 : 1 and 2 : 0.5 : 0.5) were studied, and each ratio compared with a no-transposase control. After 15 days of antibiotic selection, the number of G418-resistant colonies was determined. Every time a functional SB transposase vector was included, the number of fluorescent and G418-resistant colonies was markedly higher compared with that in the respective control without transposase (P ≤ 0.001). Interestingly, all G418-resistant colonies expressed Venus. Molecular characterisation of genomic insertions in 6 monoclonal cell lines was performed by PCR and splinkerette PCR. PCR analysis confirmed presence of the Venus transgene in all cell lines. Splinkerette PCR results revealed at least 15 transposase-catalyzed genomic insertions of the transgene. Individual cells from a polyclonal SB transgenic fibroblast culture were used as nuclear donors to produce zona-free SCNT embryos. Of the reconstructed embryos, 33% reached blastocyst stage and about half of them expressed Venus. In conclusion, SB transposase is able to actively transpose monomeric copies of transgenes into the genome of bovine cells, which can be reprogrammed upon nuclear transfer to generate morphologically normal embryos expressing the transgene of interest.

Telomeres play an important role in aging, and are critical for the regenerative capacity of mammalian cells. The holoenzyme telomerase rebuilds telomeres and is composed of two components, the catalytic protein telomerase reverse transcriptase (TERT) and the telomerase RNA (TERC). TERC is ubiquitously expressed in somatic cells and is thought to have no regulatory effects on telomerase activity. Transgenic expression of human TERT (hTERT) in bovine somatic and embryonic cells extends telomere length and enhances telomerase activity. To obtain further insight into the regulatory capacity of the two telomerase components, we have studied the ability of hTERC and hTERT to increase telomerase activity and telomere length in bovine embryos. Expression plasmids for the human RNA component (hTERC) and/or the catalytic subunit of human telomerase (hTERT), respectively, were injected into the cytoplasm of in vitro-produced bovine zygotes. Ectopic expression of hTERC increased telomerase activity and telomere length in bovine blastocysts. Coexpression of hTERT and hTERC did not result in further telomere elongation when compared to the hTERC group. These data indicate that TERC is one of the limiting factors of telomerase activity in bovine blastocysts, and further establish bovine preimplantation embryos as a useful model to modulate telomere length with impact for basic embryology and derivation of pluripotent cells.

Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.

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.

Induced pluripotent stem (iPS) cells are a seminal breakthrough in stem cell research and are promising for the development of advanced regenerative therapies and farm animal biotechnology. Considering the potential of this technology for both basic and clinical research, it is tempting to extend this research to important livestock species, such as cattle, in which authentic embryonic stem cell lines are yet not available. The first attempts to produce iPS cells from livestock species were made using retro- and lentiviral vectors, which are associated with an increased risk of insertional mutagenesis and which are not removable after reprogramming. Here, we describe a nonviral method for the derivation of bovine iPS cells, employing a piggyBac (PB) transposon system. The reprogramming PB transposon encodes the primate cDNA of 6 core reprogramming factors, OCT4, SOX2, KLF4, MYC, LIN28, and NANOG, separated by self-cleaving 2A peptide sequences and driven by the chimeric CAGGS promoter. The derived bovine iPS line expressed typical endogenous genes (OCT4, SOX2, c-MYC, KLF4, NANOG, REX1, and ALP) by RT-PCR and OCT-4 as well as SSEA-1 and 4 pluripotency-related markers by immunostaining, and it exhibited silencing of exogenous reprograming factors. Moreover, the iPS line showed long-term proliferation (until the 40th passage) under feeder-free culture conditions, differentiated into derivatives of the 3 germ layers in vitro, and formed teratomas (4/6) after subcutaneous injection into immunodeficient nude mice. These results are a major step towards the derivation of authentic bovine iPS cells, and thus facilitate the genetic modifications of the bovine genome.

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the Sleeping Beauty (SB) transposon system was assessed for stable gene transfer into the cattle genome. The transposon plasmids encoded a ubiquitously active CAGGS promoter-driven Venus reporter and a lens-specific α A-crystallin promoter driven tdTomato fluorophore, respectively. The helper plasmid carried the hyperactive SB100x transposase variant. In total, 50 in vitro-derived zygotes were co-injected (Garrels et al. 2011 PLoS ONE 6; Ivics et al. 2014 Nat. Protoc. 9) and cultured up to blastocyst stage (Day 8). Two blastocysts were Venus-positive and were transferred to synchronized heifers, resulting in one pregnancy. The resulting calf was normally developed and vital; however, it died shortly after cesarean section due to spontaneous bleeding from an undetected aneurism. Phenotypic analysis suggested that the calf was indeed double-transgenic, showing widespread expression of Venus and lens-specific expression of tdTomato. Genotyping and molecular analyses confirmed the integration of both reporter transposons and the faithful promoter-dependent expression patterns. Subdermal tissue of an ear biopsy was used to culture fibroblasts, which were employed in somatic cell nuclear transfer experiments. In total, 39 embryos were reconstructed, of which 34 underwent cleavage, and at the end of culture 12 morulas and 12 blastocysts were obtained. Ten of the blastocysts were Venus positive, and embryo transfer of Venus-positive blastocysts is planned. In summary, we showed that the cytoplasmic injection of SB components is a highly efficient method for transgenesis in cattle. Due to the modular composition of SB plasmids, even double transgenic cattle can be generated in a one-step procedure. Importantly, the SB-catalyzed integration seems to favour transcriptionally permissive loci in the genome, resulting in faithful and robust promoter-dependent expression of the transgenes. The transposon constructs carry heterospecific loxP sites, which will be instrumental for targeted insertion of functional transgenes by Cre recombinase-mediated cassette exchange.Financial support of DFG (Ku 1586/3-1), UNRC, CONICET and Agencia Nacional de Promoción Científica y Tecnológica de la Argentina (ANPCyT) is gratefully acknowledged.

Farm animals have a long-standing tradition as models in transplantation medicine. The implementation of techniques for precision genetic engineering techniques in pigs will allow to assessing safety and efficacy of novel cell therapies in this large animal model (Garrels et al. 2011 PLoS One 6, e235731). Recently, we generated a syngenic clone cohort of Sleeping Beauty (SB) transposon transgenic pigs by active transgenesis. Eight syngenic pigs carried a monomeric transposon with a Venus cDNA, and 4 animals carried a transposon with an mCherry cDNA; both groups of animals had the same genetic background and differed only in regard to the reporter transgene. In addition, half siblings resulting from conventional breeding were used as control animals (heterologous). To assess immunocompatibility within the clone cohort, skin transplantation between mCherry pigs and Venus pigs was performed. The donors (2 mCherry pigs) and the recipients (3 Venus pigs) were anesthetized and 25-mm2 patches of skin were transplanted under sterile conditions. As control, skin patches of half siblings (heterologous) were transplanted to the Venus recipients. Each recipient was transplanted with 6 small skin patches. The fluorescence reporters Venus and mCherry allowed for unambiguous tracking of cell origin, potential cell fusion, or cell migration in the transplants. Skin patches, which were transplanted between animals of the clone cohort, were accepted within 2 to 3 weeks (n = 14), whereas heterologous skin transplants (n = 4) were rejected. Some of the skin transplants were recovered for fluorescence microscopic analysis after 3 to 6 weeks. Fluorescence microscopy allowed unequivocal discrimination between host cells and cells derived from the transplant in the “wound” zone. Major advantages of this approach are the identical genetic background, omission of an immunosuppressive regime, and direct visual discrimination of transplanted and host cells by different fluorophore reporters. Importantly, no signs of transgene silencing were found for the monomeric SB transposon constructs. These data show that precision genetic engineering is possible in the domestic pig, and that the mCherry and Venus reporters are suitable for long-term cell tracking in transplantation situations. This approach is promising for the development of a preclinical model for novel cell therapies.

Recently, we produced 2 founder boars with a non-autonomous Sleeping Beauty (SB) system carrying 3 monomeric integrations of a Venus transposon cassette and showing transgene segregation during meiosis (Garrels et al. 2011 PLoS One 6, e27563). It was possible to show transmission of the reporter protein to fertilized oocytes by confocal microscopy. The aim of this study was to assess the suitability of different fluorophore reporters for in vivo labelling of pig spermatozoa. Therefore, we used Venus transposon fibroblasts from a F1 boar, which carry a single integration of the transposon cassette and used these fibroblasts for a Cre-mediated cassette exchange against an mCherry reporter. These cells were used for somatic cell nuclear transfer (SCNT) to derive a syngene clone cohort of boars, which differ only in the fluorophore reporter cDNAs (either Venus or mCherry). Importantly, this methodology did not request any antibiotic selection cassette and allows precise genetic modifications in a livestock species where no authentic embryonic stem cells are available (Garrels et al. 2012 Trends in Biotechnology 30, 386–393). A total of 8 male piglets carrying the Venus transposon, and 4 male piglets carrying the mCherry reporter were born. Three Venus boars and 2 mCherry boars were raised to sexual maturity, and ejaculated sperm was obtained with the help of a phantom. A detailed flow cytometric analysis revealed that the spermatozoa samples were specifically Venus or mCherry positive [Gallios, Beckmann Coulter, Krefeld, Germany; solid-state laser (488 nm; 22 mW), filter for green fluorescence (525 BP); filter for red fluorescence: (620/30)], respectively. In direct comparative measurements, the spermatozoa samples from transgenic boars (Venus and Cherry) and wildtype controls could be discriminated. Interestingly, spermatozoa were uniformly Venus- or mCherry-positive and gave a distinct fluorescence peak in flow-cytometric measurements. The monomeric transgenes were transmitted through the germ line according to Mendelian rules with the expected ratio of 50% transgenic and 50% nontransgenic piglets. Fluorescence microscopic analysis and Western blotting confirmed the uniform presence of Venus and mCherry in boar spermatozoa, respectively. This is the first characterisation of spermatozoa from a pig cohort carrying a targeted cassette exchange. This large animal model may help to elucidate the function of paternally transmitted components to fertilized oocytes.

Because of their antibacterial properties, silver nanoparticles (AgNP) are used abundantly in medical products such as wound dressing and catheters, but also in consumer items like clothing and lining of food containers. Despite such widespread exposure and the fact that they are considered toxic to eukaryotic cells as well, knowledge concerning their reprotoxic potential is very limited. Therefore, the aim of the present study was to investigate the effects of AgNP on embryo development. The used AgNP were produced by laser ablation in liquids, which provides particles of high purity and stability. In order to ensure direct contact of the particle with the developing organism, 10 pL of an AgNP-dispersion in water (250 µM Ag; average particle diameter 15 nm) was microinjected into one blastomere of a two-cell-stage embryo (n = 91), derived from superovulated NMRI-mice. As controls, embryos were injected with water only (n = 74) or left untreated as a handling control (n = 102). Subsequently, in vitro culture commenced for 72 hours at 37°C and 5% CO2 in KSOM plus 1% BSA. In order to distinguish whether possible effects are caused by the nanoparticles as such or by Ag+-ions released from the nanoparticles, additional embryos were co-incubated with silver nitrate (25 µM) during culture (n = 41). To exclude influence of the NO–-ions on the embryo development, potassium nitrate controls (25 µM) were run as well (n = 40). The obtained blastocyst rates were compared using one-way ANOVA and a chi-square test. Day 4 blastocysts derived from the injected groups and the handling control were used for a real-time PCR analysis, to investigate gene expression of developmentally important genes (Bax, Bcl2l2, and Tp53 for apoptosis; Oct4 and Nanog for stemness, Dnmt3a for methylation). Embryo development was assessed on a daily basis. In the injected embryos, no abnormal development was observed. After injection with silver nanoparticles, the development rate reached 61.5%, which did not differ from the water-injected embryos, where 66.2% of the embryos developed to blastocysts. The handling controls reached a significantly higher blastocyst rate of 79.4% compared to both injected groups (P

Recently, we generated transposon-transgenic boars (Sus scrofa), which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring). The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.

The pig is an important model for biomedical research. Recently, we described a method for producing transgenic pigs using a nonautonomous Sleeping Beauty (SB) transposon1 (Garrels et al. 2010 Reprod. Domest. Anim. 45, 65 abst.). Briefly, in vivo developed porcine zygotes were co-injected with a CAGGS-Venus transposon and hyperactive SB100. A total of 141 in vivo developed zygotes were injected and transferred to synchronized foster sows. Subsequent analysis revealed specific transposase-mediated integration of 1 to 5 copies of the Venus transposon in fetuses and piglets. This method results in highly efficient SB-mediated transgene transposition into the porcine genome: 57% of the fetuses examined and 42% of piglets were transgenic, representing 6.4% of the treated zygotes. The piglets showed persistent expression of the Venus reporter. Here, we present cellular analysis of 2 founder boars. Expression of the Venus reporter was observed in skin, cultured fibroblasts, leukocytes, and spermatozoa of both animals. However, flow cytometric measurement of leukocytes and cultured ear fibroblasts revealed that these boars carried both a Venus-fluorescence-positive population and a Venus-fluorescence-negative cell population. PCR analysis revealed that the Venus-fluorescence-negative cells were genotypically negative, indicating transgene mosaicism. Interestingly, all spermatozoa tested were Venus-positive and gave a distinct fluorescence peak in repeated flow-cytometric measurements (n = 6). Fluorescence microscopy revealed localisation of the Venus fluorophore in the sperm tail, in the midpiece, and in the equatorial segment of the sperm head. Motility of the transgenic sperm as measured by computer-assisted sperm analysis (Hamilton-Thorne, Beverly, MA, USA) indicated no decrease in percentage of motile sperm and the movement patterns. Sorting Hoechst 33342–stained transgenic sperm into X- and Y-chromosome bearing populations did not reveal any differences in Venus fluorescence between these 2 groups. To test the fertility of the transgenic sperm, 6 wild-type sows were artificially inseminated. Four pregnancies were established, 2 of these sows were sacrificed on Day 29 of gestation and a total of 9 Venus-positive normally developed fetuses and 2 degenerated fetuses were recovered. The other 2 pregnancies are ongoing at the time of writing. This is the first characterisation of spermatozoa from transposon transgenic pigs. The results show that Venus transposon–bearing transgenic spermatozoa are fertile and demonstrate germline transmission to the F1 offspring. Sleeping Beauty-mediated transposition is thus a promising approach for genetic modification of the pig genome.

Novel techniques such as ultrashort pulsed laser technology to produce in situ bio-conjugated nanoparticles (NP) as biomarkers for optical imaging and intervention hold great potential to observe dynamic processes in gametes and embryos without affecting their developmental potential. With regard to toxicology, current preliminary evidence is mainly based on specific cell lines and suggests low toxicity for gold NP (GNP), but a higher toxicity for silver NP (SNP), which also possess a considerable antibacterial effect. Little is known about their impact on sensitive biological systems as early mammalian embryos. The present study investigated the potential toxicity of GNP and SNP in murine embryos for the first time. Since the NP were laser-generated, they formed a stable dispersion in water without the need of surface coating, which might have caused additional toxic effects. Approximately 10 pL of a NP díspersion (average NP diameter of 15 nm) containing 50 μg mL-1 of either GNP or SNP were injected into 1 blastomere of 2-cell-stage embryos (n = 93 or 75, respectively), derived from superovulated NMRI mice. Embryos injected with water alone (n = 79) served as controls. Untreated embryos (n = 92) were run as a handling control. Successful NP injection was confirmed using laser scanning confocal microscopy. After treatment, embryos were cultured for 72 h at 37°C and 5% CO2 in KSOM plus 1% BSA. Embryo development was assessed on a daily basis. No abnormal development was observed. The handling controls reached a blastocyst rate of 77.2%. A total of 66.2% of the water-injected embryos developed to blastocysts, compared to 62.4% and 56.0% after injection of GNP and SNP, respectively. Neither 1-way ANOVA nor an additional chi-square test indicated significant differences between treatment groups. In conclusion, these preliminary data indicate that intracytoplasmatic injection of GNP and SNP had no significant impact on embryo development. Further tests, including qRT-PCR of development-relevant genes in blastocysts and transfer of injected embryos into recipient animals to study potential long-term effects, are underway to gain a better understanding of GNP and SNP embryo toxicology. We gratefully acknowledge the support of the Masterrind GmbH Verden and the NBank Niedersachsen.

Telomeres are repetitive, noncoding sequences at the ends of linear chromosomes that shorten with each cell division. They play an important role in aging and affect the regenerative capacity of cells. The holoenzyme telomerase rebuilds telomeres and is composed of 2 components, i.e. the catalytic protein component telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC). In mammals, telomerase is active during embryogenesis, in germ cells and a subset of stem and progenitor cells. In the present study, we set out to express the TERC component alone and then in combination with TERT, the human telomerase complex, in bovine embryos. The human telomerase components are highly homologous to bovine telomerase genes. Here, 3 different expression constructs encoding hTERC, hTERT, and a green fluorescent protein (GFP) reporter were co-injected into bovine zygotes cytoplasm, and three groups of 528, 1865, and 110 zygotes were constituted; hTERC/GFP (Group 1), hTERT/hTERC/GFP (Group 2), and GFP alone (Group 3), respectively. GFP fluorescence was used to identify successfully injected embryos. This method has recently been established in our laboratory. Injected and control embryos were cultured for 7 days to the blastocyst stage in vitro and the impact on early embryonic development and the physiological consequences of an ectopic overexpression of telomerase in early bovine embryos were assayed. We obtained 45 blastocysts with green fluorescence in the first, 192 in the second, and 28 in the third group. Embryos with GFP fluorescence were frozen for subsequent PCR analysis and telomerase activity measurement. Some blastocyts were analyzed using quantitative fluoresence in situ hybridization to monitor telomere length. Control groups were analyzed for the endogenous levels of TERC and TERT. Results indicate that endogenous TERC and TERT are up-regulated in morulae and blastocyts. In this study, we show that human TERC and TERT can be expressed in blastocysts by cytoplasmic plasmid injection in bovine zygotes. Statistical analyses were performed using the JMP 7.0.1 for Windows software (SAS Institute Inc., Cary, NC, USA). The Tukey-Kramer test was applied to compare the group means. The expression of hTERC alone resulted in a significant extension of telomere length of 280 telomere fluorescence units. Expression of both components also resulted in a significant extension of telomere length. In conclusion, TERC component alone is sufficient to elongate telomere length. The activity measurement showed that telomerase activity in the hTERT and hTERC injected group is 1.77 times higher than in the control group. Findings from this study will allow a comprehensive analysis of the functions of TERT and TERC in early embryogenesis. The ectopic expression of telomerase components in bovine embryos could pave new avenues for generating stem cells and for the development of novel regenerative therapies. Funded by DFG.