Eric M. Walters

Significant Improvement in Cloning Efficiency of an Inbred Miniature Pig by Histone Deacetylase Inhibitor Treatment after Somatic Cell Nuclear Transfer

The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.

Use of the CRISPR/Cas9 System to Produce Genetically Engineered Pigs from In Vitro-Derived Oocytes and Embryos

ABSTRACT Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.

Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos.

Faulty epigenetic reprogramming of somatic nuclei is likely to be a major cause of low success observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been demonstrated that the developmental competence of SCNT embryos in several species were significantly enhanced via treatment of histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) to increase histone acetylation. Here we report that 50 nM TSA for 10 h after activation increased the developmental competence of porcine SCNT embryos constructed from Landrace fetal fibroblast cells (FFCs) in vitro and in vivo, but not at higher concentrations. Therefore, we optimized the application of another novel HDACi, Scriptaid, for development of porcine SCNT embryos. We found that treatment with 500 nM Scriptaid significantly enhanced the development SCNT embryos to the blastocyst stage when outbred Landrace FFCs and ear fibroblast cells (EFCs) were used as donors compared to the untreated group. Scriptaid increased the overall cloning efficiency from 0.4% (untreated group) to 1.6% for Landrace FFCs and 0 to 3.7% for Landrace EFCs. Moreover, treatment of SCNT embryos with Scriptaid improved the histone acetylation on Histone H4 at lysine 8 (AcH4K8) in a pattern similar to that of the in vitro fertilized (IVF) embryos.

Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs

We report using zinc finger nucleases (ZFNs) and somatic cell nuclear transfer (SCNT) to generate pigs with a knockout (KO) mutation of an enhanced green fluorescent protein (eGFP) transgene. ZFNs are synthetic modular proteins composed of a FokI endonuclease domain linked to a sequence-specific zinc finger DNA-binding domain. Pairs of ZFNs bind to the target region, allowing FokI dimerization and subsequent DNA cleavage. Mutation of an eGFP transgene by using ZFNs has been demonstrated in rats (Geurts et al., 2009); however, there are no reports of ZFN-mutated livestock offspring. Using ZFNs to increase the efficiency of gene modification may advance the production of agriculturally and clinically relevant animal models, particularly in species where modification is difficult. All animal procedures followed an approved IACUC protocol. Adult porcine ear fibroblasts hemizygous for the eGFP transgene (Whitworth et al., 2009), from a grandson of the founder animal, were cultured to 75% confluency, trypsinized, and cotransfected (Ross et al., 2010) with a pair of ZFN plasmids that bind to opposing strands at the eGFP target site (Sigma–Aldrich CompoZr®) and a red fluorescent CAG-tomato plasmid as a transient selectable fluorophore reporting transfection efficiency. Transfected fibroblasts were cultured for 96 h and selected for red fluorescence by automated cell sorting (FACS; Fig. 1A,B) and seeded into 96-well plates (100 cells/well) based on CAG-tomato expression (2% of total cells sorted). A second round of FACS using negative selection of eGFP fluorescence enriched for eGFP KO cells (~5% of sorted cells). PCR of genomic DNA from fibroblasts in wells containing predominantly non-green cells was used to amplify a fragment bracketing the ZFN targeting site. The PCR product was cloned into E. coli, and 16 colonies showed mutations by Sanger sequencing. These mutations at the ZFN cleavage site included a 6-bp deletion, a 333-bp deletion, and a 222-bp deletion replaced with a 113-bp inversion of the deleted sequence (Supplemental Information http://animalsciences.missouri.edu/faculty/prather/). Fibroblast colonies determined to carry ZFN-induced mutations were used as donor cells for SCNT, and embryo transfer (n = 3; Whitworth et al., 2009). One Day-12 embryo collection recovered 7 of 9 embryos that did not fluoresce. Quality sequence was obtained from four embryos, and confirmed that the two embryos that fluoresced had an intact eGFP and two embryos that did not fluoresce were mutated. One of seven piglets recovered via Cesarean section at term for the other two pregnancies fluoresced (Fig. 1C). DNA sequencing confirmed that this piglet had unaltered sequence at the predicted ZFN cut site, while the non-fluorescing piglets had various deletions and insertions. While a direct comparison to a 12% mutation rate in the rat made by microinjection (Geurts et al., 2009) cannot be made because of our FACS preselection and SCNT methods, six out of seven founders being mutated is consistent with their results. This study establishes ZFN-based gene modification in a large animal model. The methods used are anticipated to be useful in selective knockout of endogenous swine genes of agricultural and biomedical importance without introducing any transgenic sequence into the genome. Figure 1 A: Control porcine ear fibroblasts; eGFP and Hoechst DNA stain fluorescence. B: Porcine ear fibroblasts treated with eGFP-specific ZFNs; eGFP and Hoechst DNA stain fluorescence. GFP fluorescence is absent in a high proportion of the treated fibroblasts ...

Genetically Engineered Pig Models for Human Diseases

Although pigs are used widely as models of human disease, their utility as models has been enhanced by genetic engineering. Initially, transgenes were added randomly to the genome, but with the application of homologous recombination, zinc finger nucleases, and transcription activator-like effector nuclease (TALEN) technologies, now most any genetic change that can be envisioned can be completed. To date these genetic modifications have resulted in animals that have the potential to provide new insights into human diseases for which a good animal model did not exist previously. These new animal models should provide the preclinical data for treatments that are developed for diseases such as Alzheimers disease, cystic fibrosis, retinitis pigmentosa, spinal muscular atrophy, diabetes, and organ failure. These new models will help to uncover aspects and treatments of these diseases that were otherwise unattainable. The focus of this review is to describe genetically engineered pigs that have resulted in models of human diseases.

Generation of an Inbred Miniature Pig Model of Retinitis Pigmentosa

PURPOSE The Pro23His (P23H) rhodopsin (RHO) mutation underlies the most common form of human autosomal dominant retinitis pigmentosa (adRP). The objective of this investigation was to establish a transgenic miniature swine model of RP using the human P23H RHO gene. METHODS Somatic cell nuclear transfer (SCNT) was used to create transgenic miniature pigs that expressed the human P23H RHO mutation. From these experiments, six transgenic founders were identified whose retinal function was studied with full-field electroretinography (ffERG) from 3 months through 2 years. Progeny from one founder were generated and genotyped to determine transgene inheritance pattern. Retinal mRNA was isolated, and the ratio of P23H to wild-type pig RHO was measured. RESULTS A single transgene integration site was observed for five of the six founders. All founders had abnormal scotopic and photopic ffERGs after 3 months. The severity of the ffERG phenotype was grouped into moderately and severely affected groups. Offspring of one founder inherited the transgene as an autosomal dominant mutation. mRNA analyses demonstrated that approximately 80% of total RHO was mutant P23H. CONCLUSIONS Expression of the human RHO P23H transgene in the retina creates a miniature swine model with an inheritance pattern and retinal function that mimics adRP. This large-animal model can serve as a novel tool for the study of the pathogenesis and therapeutic intervention in the most common form of adRP.

Osteopontin Reduces Polyspermy During In Vitro Fertilization of Porcine Oocytes

Abstract This study was designed to determine the role of osteopontin (SPP1) in in vitro fertilization (IVF) in swine. The initial objective was to evaluate the effect of various concentrations of SPP1 (0, 0.001, 0.01, 0.1 and 1 μg/ml) on spermatozoa and oocytes during IVF. The results demonstrate that SPP1 reduced the rate of polyspermy in a dose-dependent manner (P < 0.05). SPP1 also reduced both the number of sperm in oocytes as compared to the control and the number of spermatozoa bound to the zona pellucida (ZP) (P < 0.05). High doses of SPP1 (1 μg/ml) reduced penetration and male pronucleus formation as compared to the control (P < 0.05). Interestingly, compared to the control group, medium doses of SPP1 increased fertilization efficiency (42.6% and 44.6% vs. 31.6%; P < 0.05), representing a 41% improvement for 0.1 μg/ml SPP1). The ZP of 0.1 μg/ml SPP1-treated oocytes was more difficult to digest than control oocytes (P < 0.05). The percentage of acrosome-reacted spermato zoa bound to the ZP during IVF increased after 4 h of 1.0 μg/ml SPP1 treatment compared to 0 or 0.1 μg/ml SPP1. SPP1 did not have an effect on sperm motility, progressive motility, and sperm viability. To confirm that the reduction of polyspermy was specific to SPP1, a mixture of pregnancy-associated glycoproteins was included in the IVF protocol and shown to have no effect on polyspermy. Furthermore, Western blotting demonstrated that a 50-kDa SPP1 form was present in the oviducts on Days 0, 3, and 5 in pregnant and nonpregnant gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5. The current study represents the first report to demonstrate that SPP1 plays an important role in the regulation of pig polyspermic fertilization; it decreases polyspermy and increases fertilization efficiency during IVF.

Completion of the swine genome will simplify the production of swine as a large animal biomedical model

BackgroundAnatomic and physiological similarities to the human make swine an excellent large animal model for human health and disease.MethodsCloning from a modified somatic cell, which can be determined in cells prior to making the animal, is the only method available for the production of targeted modifications in swine.ResultsSince some strains of swine are similar in size to humans, technologies that have been developed for swine can be readily adapted to humans and vice versa. Here the importance of swine as a biomedical model, current technologies to produce genetically enhanced swine, current biomedical models, and how the completion of the swine genome will promote swine as a biomedical model are discussed.ConclusionsThe completion of the swine genome will enhance the continued use and development of swine as models of human health, syndromes and conditions.

Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency

Significance Pigs have many features that make them attractive as biomedical models, especially in regenerative medicine. Here, we have introduced inactivating mutations simultaneously into both alleles of the recombination activating gene (RAG) 2 gene in fibroblasts derived from minipigs and then used somatic-cell nuclear transfer to produce RAG2−/− cloned animals with a severe immune deficiency (SCID) phenotype and lacking T and B cells. When human induced pluripotent (iPS) cells were injected into these SCID pigs, the animals readily form teratomas representing a wide range of human tissues. Provided they can be protected from pathogens, these genetically engineered pigs could be a valuable resource as models for human patients with analogous immunodeficiencies and for testing the safety and regenerative capacity of grafts derived from iPS cells. Pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation, and tumor development and will aid in developing therapies for human SCID patients. Using a reporter-guided transcription activator-like effector nuclease (TALEN) system, we generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. Somatic-cell nuclear transfer performed with the mutated cells produced pigs with RAG2 mutations without integrated exogenous DNA. Biallelically modified pigs either lacked a thymus or had one that was underdeveloped. Their splenic white pulp lacked B and T cells. Under a conventional housing environment, the biallelic RAG2 mutants manifested a “failure to thrive” phenotype, with signs of inflammation and apoptosis in the spleen compared with age-matched wild-type animals by the time they were 4 wk of age. Pigs raised in a clean environment were healthier and, following injection of human induced pluripotent stem cells (iPSCs), quickly developed mature teratomas representing all three germ layers. The pigs also tolerated grafts of allogeneic porcine trophoblast stem cells. These SCID pigs should have a variety of uses in transplantation biology.

The cryobiology of spermatozoa

The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm cryobiology, provide a synopsis of the development of sperm cryobiology as a discipline, and present the current state and directions for future research in sperm cryobiology in the three major areas outlined above-agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.