Porcine Fetal Fibroblast Cells Research Articles

Chromatin profiling and state predictions reveal insights into epigenetic regulation during early porcine development

BackgroundGiven their physiological similarities to humans, pigs are increasingly used as model organisms in human-oriented biomedical studies. Additionally, their value to animal agriculture across the globe has led to the development of numerous studies to investigate how to improve livestock welfare and production efficiency. As such, pigs are uniquely poised as compelling models that can yield findings with potential implications in both human and animal contexts. Despite this, many gaps remain in our knowledge about the foundational mechanisms that govern gene expression in swine across different developmental stages, particularly in early development. To address some of these gaps, we profiled the histone marks H3K4me3, H3K27ac, and H3K27me3 and the SWI/SNF central ATPase BRG1 in two porcine cell lines representing discrete early developmental time points and used the resulting information to construct predicted chromatin state maps for these cells. We combined this approach with analysis of publicly available RNA-seq data to examine the relationship between epigenetic status and gene expression in these cell types.ResultsIn porcine fetal fibroblast (PFF) and trophectoderm cells (PTr2), we saw expected patterns of enrichment for each of the profiled epigenetic features relative to specific genomic regions. H3K4me3 was primarily enriched at and around global gene promoters, H3K27ac was enriched in promoter and intergenic regions, H3K27me3 had broad stretches of enrichment across the genome and narrower enrichment patterns in and around the promoter regions of some genes, and BRG1 primarily had detectable enrichment at and around promoter regions and in intergenic stretches, with many instances of H3K27ac co-enrichment. We used this information to perform genome-wide chromatin state predictions for 10 different states using ChromHMM. Using the predicted chromatin state maps, we identified a subset of genomic regions marked by broad H3K4me3 enrichment, and annotation of these regions revealed that they were highly associated with essential developmental processes and consisted largely of expressed genes. We then compared the identities of the genes marked by these regions to genes identified as cell-type-specific using transcriptome data and saw that a subset of broad H3K4me3-marked genes was also specifically expressed in either PFF or PTr2 cells.ConclusionsThese findings enhance our understanding of the epigenetic landscape present in early swine development and provide insight into how variabilities in chromatin state are linked to cell identity. Furthermore, this data captures foundational epigenetic details in two valuable porcine cell lines and contributes to the growing body of knowledge surrounding the epigenetic landscape in this species.

Establishment of PIN1 knockout porcine fetal fibroblast cell lines via CRISPR/Cas9 mediated gene-targeting

Establishment of PIN1 knockout porcine fetal fibroblast cell lines via CRISPR/Cas9 mediated gene-targeting

Improving porcine SCNT efficiency by selecting donor cells size

ABSTRACT Considerable advancements have recently been achieved in porcine somatic cell nuclear transfer (SCNT), but the efficiency remains low. Donor cell size might play an important role in SCNT, but its effects in pigs remain unclear. This study aimed to evaluate the efficiency of porcine SCNT by selecting donor cells of suitable size. Porcine fetal fibroblasts (PFFs) were divided into three groups, group S (small, d ≤ 13 μm), group M (medium, 13 μm<d ≤ 18 μm), and group L (large, d > 18 μm), and their biological characteristics were analyzed. Next, SCNT was performed using PFFs of different sizes to evaluate the developmental potential of reconstructed embryos. The data showed that PFFs in groups S, M and L accounted for 17.5%, 47.7% and 34.8% of cells, respectively. Morphologically, cells in group S exhibited clear and regular cell membranes and nuclei, whereas cells in groups M and L displayed varying degrees of cell membrane protuberance, karyo-pyknosis, autophagy and mitochondrial abnormalities. In addition, the growth status and proliferation capabilities of cells in group S were significantly better than those of group M and group L. The percentage of cells at G0/G1 in group S and M were significantly greater than group L. The senescence rate of group S was lower than group M and group L. The apoptosis rate of group S was significantly lower than that of group L but comparable to that of group M . The cleavage rate of group S was also significantly greater than that of group M but comparable to that of group L . The blastocyst rate of group S was significantly greater than that of group M and group L. The blastocyst cell numbers of group S were also significantly greater than those of group M and group L. These findings suggested that small PFFs with a diameter of less than 13 μm are more suitable donor cells for SCNT in pigs. Abbreviations: DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; PFFs: Porcine fetal fibroblast cells; SCNT: Somatic cell nuclear transfer.

Effect of ARTEMIS (DCLRE1C) deficiency and microinjection timing on editing efficiency during somatic cell nuclear transfer and in vitro fertilization using the CRISPR/Cas9 system

The ability to efficiently introduce site-specific genetic modifications to the mammalian genome has been dramatically improved with the use of the CRISPR/Cas9 system. CRISPR/Cas9 is a powerful tool used to generate genetic modifications by causing double-strand breaks (DSBs) in DNA. Artemis (ART; also known as DCLRE1C), is a nuclear protein and is essential for DSB end joining in DNA repair via the canonical non-homologous end joining (c-NHEJ) pathway. In this work, we tested whether ART deficiency affects DNA repair following CRISPR/Cas9 induced DSBs in somatic cells. We also demonstrated the effect of microinjection timing on embryo developmental ability and gene targeting efficiency of CRISPR/Cas9 system to disrupt the interleukin 2 receptor subunit gamma (IL2RG) locus using porcine in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryos. In comparison to non-injected controls, CRISPR/Cas9 injection of IVF derived zygotes at 4 h and 8 h after fertilization did not impact cleavage and blastocyst rate. Gene modification rate was observed to be higher, 53.3% (9/16) in blastocysts injected 4 h post-fertilization as compared to 11.1% (1/9) in blastocysts injected 8 h post-fertilization. Microinjection 8 h after chemical activation of SCNT derived embryos decreased blastocyst development rate compared to non-injected controls but showed a higher gene modification efficiency of 66.7% as compared to 25% in the 4 h post-activation injection group. Furthermore, we observed that male ART-/- and ART+/- porcine fetal fibroblast (pFF) cells showed lower modification rates (2.5% and 1.9%, respectively) as compared to the ART intact cell line (8.3%). Interestingly, the female ART-/- and ART+/- pFF cells had modification rates (4.2% and 10.1%, respectively) similar to those seen in the ART intact cells. This study demonstrates the complex effect of various parameters such as microinjection timing and ART deficiency on gene editing efficiency in in vitro derived porcine embryos.

Reprogramming Porcine Fibroblast to EPSCs.

The development of porcine expanded potential stem cells (pEPSCs) provides an invaluable tool for investigation of porcine stem cell pluripotency and opens a venue for research in biotechnology, agriculture, and regenerative medicine. Since the derivation of pEPSC from porcine pre-implantation embryos has been demanding in resource supply and technical challenges, it is more feasible and convenient for most laboratories to derive this new type of porcine stem cells by reprogramming somatic cells. In this chapter, we describe the detailed procedures for reprogramming porcine fetal fibroblast cells to EPSCiPSC with the eight reprogramming factors cloned on the piggyBac vectors followed by a selection for pluripotent cells independent of transgene expression using the EPSC media. This technique allows the generation of pEPSCs for stem cell research, genome editing, biotechnology, and agriculture.

Cytosine Base Editor (hA3A-BE3-NG)-Mediated Multiple Gene Editing for Pyramid Breeding in Pigs.

Pig is an important agricultural economic animal, providing large amount of meat products. With the development of functional genomics and bioinformatics, lots of genes and functional single nucleotide polymorphisms (SNPs) related to disease resistance and (or) economic traits in pigs have been identified, which provides the targets for genetic improvement by genome editing. Base editors (BEs), combining Cas9 nickase and cytidine or adenine deaminase, achieve all four possible transition mutations (C-to-T, A-to-G, T-to-C, and G-to-A) efficiently and accurately without double strand breaks (DSBs) under the protospacer adjacent motif (PAM) sequence of NGG. However, the NGG PAM in canonical CRISPR-Cas9 can only cover approximately 8.27% in the whole genome which limits its broad application. In the current study, hA3A-BE3-NG system was constructed with the fusion of SpCas9-NG variant and hA3A-BE3 to create C-to-T conversion at NGN PAM sites efficiently. The editing efficiency and scope of hA3A-BE3-NG were confirmed in HEK293T cells and porcine fetal fibroblast (PFF) cells. Results showed that the efficiency of hA3A-BE3-NG was much higher than that of hA3A-BE3 on NGH (H = A, C, or T) PAM sites (21.27 vs. 2.81% at average). Further, nonsense and missense mutations were introduced efficiently and precisely via hA3A-BE3-NG in multiple pig economic trait-related genes (CD163, APN, MSTN, and MC4R) in PFF cells by one transfection. The current work indicates the potential applications of hA3A-BE3-NG for pyramid breeding studies in livestock.

Improved production of GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pigs for xenotransplantation by recloning.

Multiple genetic modification is necessary for successful xenotransplantation from pigs. However, multiple-genetically modified cells usually suffer from various drug selections and long-term in vitro culture, which have a poor performance for somatic cell nuclear transfer (SCNT) to produce genetically modified pigs. We used to generate GTKO/hCD55/hCD59 triple-gene modified pigs by using drug-selective cell lines for SCNT, but the majority of cloned pigs were transgenic-negative individuals. In this study, to improve the production efficiency of multiple genetically modified pigs, we performed the recloning process by using transgenic porcine fetal fibroblast cells. As a result, two fetuses expressing hCD55 and hCD59 were obtained from 12 live-cloned fetuses, and one carrying high transgene expression was selected as a source of donor cells for recloning. Then we obtained 12 cloned piglets, all GTKO and carrying hCD55 and hCD59. Both hCD55 and hCD59 were expressed in fibroblast cells, but the expression levels of hCD55 and hCD59 were different among these piglets. Furthermore, piglet P5# had the highest expression of hCD55 and hCD59 in fibroblast cells than other piglets. Correspondingly, fibroblast cells of piglet P5# had significantly higher resistance against human serum-mediated cytolysis than those of piglet P11#. In conclusion, our results firstly provide support for improving efficiency of generating multiple genetically modified pig by recloning.

Integrated analysis of miRNA and mRNA expression profiles in p53-edited PFF cells

ABSTRACT p53 is the most frequently mutated gene in human cancers, with over half of all tumors harboring mutation at this locus. R248 and R249 (corresponding to porcine R241 and R242), are among the hotspot mutations frequently mutated in liver, lung, breast, and some other cancers. In this study, p53 gene was knocked out or point-edited (R241 and R242 were converted to 241W and 242S) in porcine fetal fibroblast (PFF) cells via CRISPR-Cas9 technique. High throughput sequencing of miRNA and mRNA uncovered a total of 225 differentially expressed miRNAs (DEMs) and 738 differentially expressed genes (DEGs) in the p53 knockout (p53-KO) cells, and a total of 211 DEMs and 722 DEGs in the point-modified (p53-241W242S) cells. Totally 28 annotated DEMs were found to overlap between p53-KO/p53-WT and p53-241W242S/p53-WT miRNAs datasets, of which miR-34 c, miR-218, miR-205, miR-105-1, miR-105-2, miR-206, miR-224 and miR-429 play important roles in p53 regulatory network. Among the top 10 DEGs in p53-KO and p53-241W242S cells, most genes were reported to be involved in tumors, cell proliferation or cell migration. p53-KO and p53-241W242S cells showed a significantly higher (P < 0.01) proliferation rate compared with p53-WT cells. In conclusion, genetic modifications of p53 gene significantly affect the expression levels of a large number of genes and miRNAs in the PFF cells. The p53-edited PFF cells could be used as non-tumor cell models for investigating the p53 signaling network, and as donor cells for somatic nuclear transfer, with the aim to develop porcine models with the corresponding p53 mutations. Abbreviations: CRISPR-Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; PFF: porcine fetal fibroblasts; SCNT: somatic cell nuclear transfer; RNA sequencing: small RNA sequencing and mRNA sequencing; DEGs: differentially expressed mRNAs; DEMs: differentially expressed miRNAs.

17 Generation of myostatin gene knockout boars by somatic cell nuclear transfer

Skeletal muscle is the most economically valuable tissue part in meat-producing animals, and enhancing muscle growth in these species may increase the efficiency of meat production. Skeletal muscle mass is negatively regulated by myostatin (MSTN), and nonfunctional mutations of this MSTN gene in various animal species have led to dramatic hypermuscularity. A porcine fetal fibroblast cell line with biallelic MSTN mutations (MSTN −/−), consisting of a 2-bp deletion in one allele and a 4-bp deletion in the other allele, was used as a donor to generate cloned pigs via somatic cell nuclear transfer. Cloned embryos were transferred into seven surrogates and 38 live piglets were delivered. Using these MSTN null piglets, the detailed morphological, gene and protein expression analyses were performed not only in biceps femoris, semitendinosus, and diaphragm of MSTN null piglets but also in heart, liver, spleen, lung, kidney, and tongue. The results showed that MSTN −/− boars have visually clear hypermuscular characteristics, which was supported by the increased carcass dressing percentage and loin eye size and decreased backfat thickness. The fibre cross-sectional areas of the semitendinosus and semimembranosus muscles were much larger in MSTN −/− piglets than in wild-type (WT) piglets (98.0±8.2 vs. 62.7±4.7, 105.7±11.6 vs. 72.3±8.4mm2, respectively; P&amp;lt;0.05). MSTN −/− pigs showed a higher proportion of fast-type fibres (Type-II fibre%, 95±1.8 vs. 86±2.4; P&amp;lt;0.05) and lower succinate dehydrogenase activity in muscles than WT pigs. The mRNA expression of myosin heavy-chain IIB in both two muscles was higher in MSTN −/− pigs compared with WT pigs (P&amp;lt;0.05). In organs, the heart and liver were lighter in MSTN −/− piglets than in WT piglets (8.1±0.3 vs. 10.3±0.4, 26.9±1.8 vs. 34.2±1.2g kg−1, respectively; P&amp;lt;0.05), whereas the tongue was heavier in MSTN −/− piglets than in WT piglets and myofibres of the tongue were significantly larger in the former piglets than in the latter piglets (P&amp;lt;0.01). Messenger RNA expression of MSTN in all organs was significantly lower in MSTN −/− piglets than in WT piglets (P&amp;lt;0.01) and follistatin in the heart and liver was significantly higher in MSTN −/− piglets than in WT piglets (P&amp;lt;0.05). Protein expression of MSTN in the heart, kidneys, and tongue was significantly lower in MSTN −/− piglets than in WT piglets (P&amp;lt;0.01). In conclusion, MSTN −/− pigs showed a phenotype of supermuscular ratio is associated with increased muscle fibres and smaller internal organs and has a complicated gene expression patterns.

Screening high-quality fetal bovine serum for porcine oocyte maturation in vitro.

Fetal bovine serum (FBS) is widely used in cell cultures due to its high stability and easy access. It was also used as a substitute for porcine follicular fluid (PFF) in previous studies. However, FBS components are unclear, and the presence of FBS in culture media may introduce a variation from batch to batch. This study aimed to establish an effective method to screen FBS in place of PFF in the culture media for porcine oocytes in vitro. We screened FBS from different sources by using porcine fetal fibroblast cells. The effects of six FBS samples on porcine fetal fibroblast cell growth were tested via frozen cell survival assay, cell clone formation assay, cell growth curve, and cell passage activity assay. The best serum that we called GFBS (heat‐inactivated FBS, cat. no. 10500‐64; Gibco) showed a similar effect on the maturation and development of porcine oocytes to that of PFF and can be used as a good substitute for PFF. These results suggested that the porcine fetal fibroblast cell culture test can be used as a valuable method to screen FBS for porcine oocyte maturation and embryonic development in vitro.

Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5′- and 3′-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.

Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells

CRISPR/Cas9 is an efficient customizable nuclease to generate double-strand breaks (DSBs) in the genome. This process results in knockout of the targeted gene or knock-in of a specific DNA fragment at the targeted locus in the genome of various species. However, efficiency of knock-in mediated by homology-directed repair (HDR) pathway is substantially lower compared with the efficiency of knockout mediated by the nonhomologous end-joining (NHEJ) pathway. Suppressing NHEJ pathway or enhancing HDR pathway has been proven to enhance the nuclease-mediated knock-in efficiency in cultured cells and model organisms. We here investigated the effect of small molecules, Scr7, L755507 and resveratrol, on promoting HDR efficiency in porcine fetal fibroblasts. Results from eGFP reporter assay showed that these small molecules could increase the HDR efficiency by 2–3-fold in porcine fetal fibroblasts. When transfecting with the homologous template DNA and CRISPR/Cas9 plasmid and treating with small molecules, the rate of knock-in porcine fetal fibroblast cell lines with large DNA fragment integration could reach more than 50% of the screened cell colonies, compared with 26.1% knock-in cell lines in the DMSO-treated group. The application of small molecules offers a beneficial approach to improve the frequency of precise genetic modifications in primary somatic cells.

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Generation of Transgenic Porcine Fibroblast Cell Lines Using Nanomagnetic Gene Delivery Vectors.

The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-016-9934-1) contains supplementary material, which is available to authorized users.

27 AN IMPROVED LARGE ANIMAL MODEL FOR THE STUDY OF ADULT STEM CELLS

Murine models for the study of adult stem cell populations have broadened the understanding of previously uncharacterized stem cell niches. The development of murine reporter lines for the leucine-rich repeat-containing G-protein-coupled receptor-5 (Lgr5) has highlighted the importance of this gene as a stem cell marker in the stomach, intestine, hair follicle, liver, and kidney in mice. These models however have significant limitations in terms of translational applications because of anatomical and physiological differences between humans and mice. In order to overcome these limitations, we have sought to develop a porcine LGR5 reporter line. We report the generation of a porcine stem cell reporter line using the combination of transcription activator-like effector nucleases and somatic cell NT. Transcription activator-like effector nuclease-mediated homologous recombination was used to drive the integration of an internal ribosome entry site green fluorescent protein fusion into the 3′ untranslated region of the LGR5 locus in porcine fetal fibroblast cells. Multiple cell lines were developed and screened for the proper integration event. Upon confirmation of proper integration by genomic DNA sequencing, these lines were used as donors for somatic cell NT. Transfer of the somatic cell NT reconstructed embryos to a surrogate gilt resulted in 3 live births, and the establishment of a founder line of LGR5-green fluorescent protein reporter pigs. We have begun to characterise these lines, having observed fluorescent labelling of putative stem cell populations in the intestinal crypts and hair follicles from these animals. Many of these observations parallel the expression patterns observed in similar murine models. We have confirmed the fluorescent reporter signal by immunohistochemistry using an anti-green fluorescent protein antibody, and are working towards colocalization studies using anti-LGR5 antibodies and RNA in situ hybridization, as well as the characterisation of additional stem cell populations in the pig. The development of this line of transgenic pigs represents significant progress toward the study of adult stem cells, their progenitors, and the stem cell niche, using a large animal model with an anatomy, physiology, and ability to recapitulate human disease that overcomes the current limitations of rodent models. Funding was provided by NIH R21OD019738.

21 GENERATION OF GGTA1 AND CMAH BIALLELIC KNOCKOUT PORCINE FIBROBLAST CELL LINES BY TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE AND CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9

The use of porcine organs for pig-to-human transplantation is considered the most promising solution to overcome the growing shortage of human grafts for all transplantation. Unfortunately, human antibodies recognise Gala-1, 3-gal (Gal), and N-glycolylneuraminic acid (Neu5Gc) epitopes on the surface of pig cells, causing hyperacute rejection. Transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are 2 powerful tools for producing gene knockouts with high efficiency. Here we generate α-1, 3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene knockout porcine fibroblasts cell lines with TALEN and CRISPR/Cas9. Porcine fibroblasts were treated with TALEN and CRISPR/Cas9 designed against the region coding for the catalytic core of GGTA1 and CMAH. A magnetic separation of these cells was used in somatic cell NT. The cloned fetuses and piglets were characterised by T7E1, fluorescent PCR, and DNA sequencing. Nineteen fetuses were harvested using TALEN; 4 of them were GGTA1 gene mutated, and 11 were CMAH gene mutated. The DNA sequence analysis confirmed 5 of 11 are biallelic disruptions of the CMAH gene. For the CRISPR/Cas9, 6 piglets were obtained, and DNA sequence analysis confirmed 1 of 6 is biallelic disruptions of the GGTA1 and 2 of 6 are mono-allelic disruptions of the CMAH gene. We produced several fetuses or piglets via gene editing technology (TALEN and CRISPR/Cas9) and somatic cell NT technology. Furthermore, GGTA1 and CMAH gene knockout porcine fetal fibroblast cell lines derived from the fetus individuals were successfully established. However, expression of α-Gal and Neu5Gc epitopes is needed to be further examined by flow cytometry and confocal microscopy. Our study will provide the possibility of making GGTA1 and CMAH knockout pigs in the future.

360. Magnetofection: A Versatile Approach for Messenger RNA Delivery

Transcript therapy, using stabilized non-immunogenic messenger RNA (SNIM RNA), overcomes the potential risks of mutagenicity and immunogenicity, normally associated with DNA based gene delivery and recombinant protein therapy, respectively.Magnetofection, as a delivery system, has been shown to enhance DNA based viral and non-viral gene delivery. In this method, viral or non-viral gene carriers containing nucleic acids are associated with magnetic nanoparticles and a gradient magnetic field is applied to pull them on to the target cell surface.In this study, we established a magnetofection based protocol for enhanced mRNA delivery, using non-viral carriers. Firstly, we compared magnetofection, using different magnetic nanoparticles, to lipofection and polyfection, to find the best protocol for in vitro mRNA delivery. Then, the established protocol was generalized for co-transfection of up to three reporter SNIM RNAs (Luc, eGFP and RFP) in mono-culture of Primary Mouse Embryonic Fibroblasts (PMEF), or co-culture of PMEF and Porcine Fetal Fibroblast (PFF). In parallel, expression kinetics post magnetofection of d2eGFP SNIM RNA were determined.Magnetofection significantly improved transfection efficiency in mono culture as well as co-culture of applied cells. Using this protocol, co-transfection of different SNIM RNAs did not inhibit the transfection of every single SNIM RNA into PMEF or PFF cells. Moreover, rapid and higher peak of expression was observed with using magnetic nanoparticles.To conclude, magnetofection of mRNA presents itself as an efficient mRNA delivery system, thereby bringing transcript therapies a step closer to their clinical uses.

31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN). Table 1.Production efficiency of gene knockout cloned pigs using genome editing This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.

Identification of the porcine sialoadhesin gene promoter region and its cell-specific expression in porcine alveolar macrophage cells.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the PRRS virus (PRRSV), is a communicable disease. PRRS caused huge economic losses to swine breeding. The porcine alveolar macrophage (PAM) cell is the main target cell of PRRSV; therefore, it is very important to identify the specific gene promoter that controls expression in PAM cells so that the anti-PRRSV exogenous gene can be efficiently and specifically expressed in PAM cells to improve porcine resistance to PRRSV. In this study, the transcription initiation site for sialoadhesin (Siglec-1), which is a porcine alveolar macrophage-specific gene, was determined by 5' rapid amplification of cDNA end, and 88 bp of the 5'-untranslated region was cloned. Siglec-1 promoter activity was detected by a dual-luciferase reporter assay, which showed that the fragment from -173 to +81 bp had the strongest promoter activity. Additionally, the cell-specific expression of the promoter fragments was tested in a PAM cell line (CRL-2844 cells), porcine kidney 15 cell line (PK-15 cells), porcine fetal fibroblast (PEF) cells, and porcine preadipocytes. These results also showed that the fragment from -173 to +81 bp had the strongest cell-specific expression in PAM cells.

Effects of 5-aza-2’-deoxyctidine on the development of porcine parthenogenetic and nuclear transfer embryos

The current study was to investigate whether embryo or fetal fibroblast cells treated with 5-aza-2’-deoxyctidine (5-aza-dC) have a positive effect on the in vitro development of porcine parthenogenetic and cloned embryos. To this end, porcine fetal fibroblast cells were treated with different concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours), the results showed that DNA methylation in PRE-1 SINE region was gradually reduced over time in cells treated with 5-aza-dC. To determine the effect of 5-aza-dC on in vitro development of porcine activated oocytes, the parthenogenetic embryo was treated with 5-aza- dC. Notably, treatment with 5 nM 5-aza-dC for 1 hour led to a significant improvement in blastocyst development, compared with the control group. The effects of donor cell treatment with 5-aza-dC on porcine cloned embryos development were further examined by treating fetal fibroblast cells with various concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours). Exposure of cells in 5 nM 5-aza-dC for 1 - 20 hours led to a significant improvement in the percentage of developed blastocysts, while treatment with 500 nM 5-aza-dC did not affect blastocyst development, compared to untreated controls. These findings indicate that treatment of fetal fibro-blast cells with relatively low concentrations of 5-aza-dC for short exposure times improves subsequent blastocyst development of porcine cloned embryos.