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Although stromal fibroblasts play a critical role in cancer progression, their identities remain unclear as they exhibit high heterogeneity and plasticity. Here, a master transcription factor (mTF) constructing core-regulatory circuitry, PRRX1, which determines the fibroblast lineage with a myofibroblastic phenotype, is identified for the fibroblast subgroup. PRRX1 orchestrates the functional drift of fibroblasts into myofibroblastic phenotype via TGF-β signaling by remodeling a super-enhancer landscape. Such reprogrammed fibroblasts have myofibroblastic functions resulting in markedly enhanced tumorigenicity and aggressiveness of cancer. PRRX1 expression in cancer-associated fibroblast (CAF) has an unfavorable prognosis in multiple cancer types. Fibroblast-specific PRRX1 depletion induces long-term and sustained complete remission of chemotherapy-resistant cancer in genetically engineered mice models. This study reveals CAF subpopulations based on super-enhancer profiles including PRRX1. Therefore, mTFs, including PRRX1, provide another opportunity for establishing a hierarchical classification system of fibroblasts and cancer treatment by targeting fibroblasts.

Background Canine cloning technology based on somatic cell nuclear transfer (SCNT) combined with genome-editing tools such as CRISPR-Cas9 can be used to correct pathogenic mutations in purebred dogs or to generate animal models of disease. Results We constructed a CRISPR-Cas9 vector targeting canine DJ-1. Genome-edited canine fibroblasts were established using vector transfection and antibiotic selection. We performed canine SCNT using genome-edited fibroblasts and successfully generated two genome-edited dogs. Both genome-edited dogs had insertion-deletion mutations at the target locus, and DJ-1 expression was either downregulated or completely repressed. Conclusion SCNT successfully produced genome-edited dogs by using the CRISPR-Cas9 system for the first time.

Simple Summary CRISPR/Cas9 driven multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to DNA double-strand breaks at multiple loci simultaneously. To overcome this problem in porcine cells we utilized Target-AID, a base editing system, to edit multiple loci in the porcine genome. We showed that the Target-AID system works well in porcine fibroblasts with up to 63.15% efficiency. This is the first report demonstrating that the Target-AID system works well in porcine cells and can be used to generate genome-edited pigs. Abstract Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

Gene integration at site-specific loci, such as safe harbor regions for stable expression via transgenesis, is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor site for human and mouse studies. In the present study, we found an AAVS1-like sequence in the porcine genome using the UCSC Genome Browser and designed TALEN and CRISPR/Cas9 to target AAVS1. The efficiency of CRISPR/Cas9 for targeting the AAVS1 locus in porcine cells was superior to that of TALEN. An AAVS1-targeting donor vector containing GFP was designed and cloned. We added a loxP-lox2272 cassette sequence to the donor vector for further exchange of various transgenes in the AAVS1-targeted cell line. The donor vector and CRISPR/Cas9 components targeting AAVS1 were transfected into a porcine fibroblast cell line. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in at the AAVS1 locus was confirmed by PCR analysis. To induce recombinase-mediated cassette exchange (RMCE), another donor vector containing the loxP-lox2272 cassette and inducible (Tet-on) Cre recombinase was cloned. The Cre-donor vector was transfected into the AAVS1-targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR analysis. In conclusion, gene targeting at the AAVS1 locus and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.

Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs.In an attempt to produce klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7 days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes.Klotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.

Background Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs. Results In an attempt to produce klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7 days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes. Conclusions Klotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.

Thyroid hormone (TH) has long been believed to play a minor role in male reproduction. However, evidences from experimental model of thyrotoxicosis or hypothyroidism suggests its role in spermatogenesis. Cellular action of TH requires membrane transport via specific transporters such as monocarboxylate transporter 8 (MCT8). SLC16A2 (encodes for MCT8) inactivating mutation in humans can lead to Allan-Herndon Dudley-syndrome, a X-linked psychomotor and growth retardation. These patients present cryptorchidism which suggests a role of MCT8 during spermatogenesis. In this study, we found that Mct8 is highly expressed during early postnatal development and decreases its expression in the adulthood of testis of wild-type male rats. Histological analysis revealed that spermatogonia largely lacks MCT8 expression while spermatocytes and maturing spermatids highly express MCT8. To further understand the role of Mct8 during spermatogenesis, we generated Slc16a2 (encodes MCT8) knockout rats using CRISPR/Cas9. Serum THs (T3 and T4) level were significantly altered in Slc16a2 knockout rats when compared to wild-type littermates during early to late postnatal development. Unlike Slc16a2 knockout mice, Slc16a2 knockout rats showed growth delay during early to late postnatal development. In adult Slc16a2 knockout rats, we observed reduced sperm motility and viability. Collectively, our data unveil a functional involvement of MCT8 in spermatogenesis, underscoring the importance of TH signaling and action during spermatogenesis.

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) system is now widely utilized to generate genome engineered mice; however, some studies raised issues related to off-target mutations with this system. Herein, we utilized the Campylobacter jejuni Cas9 (CjCas9) system to generate knockout mice. We designed sgRNAs targeting mouse Tyr or Foxn1 and microinjected into zygotes along with CjCas9 mRNA. We obtained newborn mice from the microinjected embryos and confirmed that 50% (Tyr) and 38.5% (Foxn1) of the newborn mice have biallelic mutation on the intended target sequences, indicating efficient genome targeting by CjCas9. In addition, we analyzed off-target mutations in founder mutant mice by targeted deep sequencing and whole genome sequencing. Both analyses revealed no off-target mutations at potential off-target sites predicted in silico and no unexpected random mutations in analyzed founder animals. In conclusion, the CjCas9 system can be utilized to generate genome edited mice in a precise manner., Highlights • Generate genetically engineered mice using the Campylobacter jejuni Cas9 (CjCas9) system. • CjCas9 showed reasonably high biallelic InDel mutation rate (up to 50%) in newborn mice. • CjCas9 system showed relatively higher specificity compared to SpCas9 system.

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18–38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs., Genome editing: Improving efficiency by improving DNA repair A method to improve the efficiency of repairing double-stranded DNA breaks facilitates the production of animals with edited genomes. The CRISPR/Cas9 system has been revolutionising biomedical research as it allows the insertion or deletion of any DNA sequence at specific sites by making double-strand DNA breaks. However, the insertion of precise genetic changes is limited by the relatively low efficiency of the repair mechanism, which requires a template DNA molecule to promote error-free repair of the DNA strands. A team of South Korean scientists led by Su Cheong Yeom at Seoul National University and Han Woong Lee at Yonsei University show that delivering multiple overlapping single-guide RNAs (that share at least five base pairs of the target sequence) as a DNA template significantly improves the repair and thus the production of mice with a targeted gene insertion.

Charcot-Marie-Tooth disease (CMT) is a genetic disorder that can be caused by aberrations in >80 genes. CMT has heterogeneous modes of inheritance, including autosomal dominant, autosomal recessive, X-linked dominant, and X-linked recessive. Over 95% of cases are dominantly inherited. In this study, we investigated whether regulation of a mutant allele by an allele-specific small interfering RNA (siRNA) can alleviate the demyelinating neuropathic phenotype of CMT. We designed 19 different allele-specific siRNAs for Trembler J (Tr-J) mice harboring a naturally occurring mutation (Leu16Pro) in Pmp22. Using a luciferase assay, we identified an siRNA that specifically and selectively reduced the expression level of the mutant allele and reversed the low viability of Schwann cells caused by mutant Pmp22 over-expression in vitro. The in vivo efficacy of the allele-specific siRNA was assessed by its intraperitoneal injection to postnatal day 6 of Tr-J mice. Administration of the allele-specific siRNA to Tr-J mice significantly enhanced motor function and muscle volume, as assessed by the rotarod test and magnetic resonance imaging analysis, respectively. Increases in motor nerve conduction velocity and compound muscle action potentials were also observed in the treated mice. In addition, myelination, as evidenced by toluidine blue staining and electron microscopy, was augmented in the sciatic nerves of the mice after allele-specific siRNA treatment. After validating suppression of the Pmp22 mutant allele at the mRNA level in the Schwann cells of Tr-J mice, we observed increased expression levels of myelinating proteins such as myelin basic protein and myelin protein zero. These data indicate that selective suppression of the Pmp22 mutant allele by non-viral delivery of siRNA alleviates the demyelinating neuropathic phenotypes of CMT in vivo, implicating allele-specific siRNA treatment as a potent therapeutic strategy for dominantly inherited peripheral neuropathies.

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell–derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus, however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR-Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components. Our plasmid-mediated procedure and the direct delivery of CRISPR-Cas9 RNPs can both be utilized to modulate traits of interest with high accuracy and efficiency in apple and grapevine, and could be extended to other crop species. The complete protocol employing the direct delivery of CRISPR-Cas9 RNPs takes as little as 2-3 weeks, whereas the plasmid-mediated procedure takes >3 months to regenerate plants and study the mutations.

Xenotransplantation of pig islet regarded as a good alternative to allotransplantation. However, cellular death mediated by hypoxia-reoxygenation injury after transplantation disturb success of this technique. In the present study, we produce transgenic pig expressing human heme oxygenase 1 (HO1) genes to overcome cellular death for improving efficiency of islet xenotransplantation. Particularly, Korean miniature pig breed, Micro-Pig, was used in the present study. Somatic cell nuclear transfer (SCNT) technique was used to produce the HO1 transgenic pig. Six alive transgenic piglets were produced and all the transgenic pigs were founded to have transgene in their genomic DNA and the gene was expressed in all tested organs. Also, in vitro cultured fibroblasts derived from the HO1 transgenic pig showed low reactive oxygen species level, improved cell viability and reduced apoptosis level.

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.

Non-metastatic cells 1 (NME1) or nm23 is the first metastasis suppressor gene discovered. It functions through various enzymatic activities and interacts with many intracellular proteins. The NME1 gene encodes two splicing variants, NME1 and NME1L. Most studies have focused on NME1 because of its abundance in cells. We previously reported NME1L-mediated suppression of NF-κB signaling by interacting with and inhibiting IKKβ. In this study, we demonstrated that NME1L, but not NME1, mediated inhibition of cell proliferation, although both NME1 and NME1L were involved in suppressing metastasis. A reporter gene assay was performed to determine the growth signaling pathway regulated by NME1L but none of the growth factors tested could induce an NF-κB-dependent luciferase expression except TNFα. Interestingly, SRE-reporter gene activation by IGF1 was significantly downregulated, along with reduction of ERK phosphorylation in NME1L expressing cells, compared to vector or NME1 expressing cells. NME1L directly interacted with KSR1, which is a scaffold for Raf-1, MEK, and ERK, that regulates ERK activation. Hence, NME1L plays a crucial role in regulation of cell proliferation by inhibiting IGF1-stimulated ERK phosphorylation through N-terminal 25 amino acid-mediated interaction with KSR1.

Mesenchymal stem cells (MSCs) have the ability to differentiate into multi-lineage cells, which confers great promise for use in regenerative medicine. In this study, MSCs were isolated from adipose tissue, bone marrow, ear skin, lung, and abdominal skin of miniature pigs (mpMSCs), and the optimal medium (DMEM/F12-Glutamax) was selected for the culturing of mpMSCs. As a result, proliferation of the mpMSCs derived from all tissues was steadily increased when cultured with DMEM/F12-Glutamax during 14 consecutive passages. The cells harbored MSC surface markers (CD34-, CD45-, CD29+, CD44+, CD90+, and CD105+), whose levels of expression differed among the tissue sources and declined over sub-passaging. In addition, the expression of stemness markers (Oct4, Sox2, and Nanog) and differentiation into mesoderm (adipocytes, chondrocytes, and osteoblasts) were clearly represented at early passage; however, expression of stemness markers decreased, and differentiation potential was lost over sequential sub-passaging, which should be considered in the selection of mpMSC for MSC-based application.

SummaryOctamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.

Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p 0.05). Caspase-3/-7 activity of the shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p 0.05). These results show that shTNFRI-Fc and HA-hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury. Utilization of the F2A self-cleaving peptide is a promising tool for generating multiple TG pigs for xenotransplantation.

In order to investigate genetic stability and gene expression profile after cloning procedure, two groups of cloned pigs were used for swine leukocyte antigen (SLA) gene nucleotide alteration and microarray analyses. Each group was consist of cloned pigs derived from same cell line (n=3 and 4, respectively). Six SLA loci were analyzed for cDNA sequences and protein translations. In total, 16 SLA alleles were identified and there were no evidence of SLA nucleotide alteration. All SLA sequences and protein translations were identical among the each pig in the same group. On the other hand, microarray assay was performed for profiling gene expression of the cloned pigs. In total, 43,603 genes were analyzed and 2,150～4,300 reliably hybridized spots on the each chip were selected for further analysis. Even though the cloned pigs in the same group had identical genetic background, 18.6～47.3% of analyzed genes were differentially expressed in between each cloned pigs. Furthermore, on gene clustering analysis, some cloned pigs showed abnormal physiological phenotypes such as inflammation, cancer or cardiomyopathy. We assumed that individual environmental adaption, sociality and rank in the pen might have induced these different phenotypes. In conclusion, the results of the present study indicate that SLA locus genes appear to be stable following SCNT. However, gene expressions and phenotypes between cloned pigs derived from the same cell line were not identical even under the same rearing conditions.

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 µg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 µg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 µg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 µg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.

The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.

The purpose of this study was to analyze the reproductive ability of transgenic female dogs born bysomatic cell nuclear transfer and to determine inheritance of the red fluorescent protein (RFP) transgene. The four founder transgenic bitches (F0) reached puberty at 340.8 ± 39.6 days after birth and were bred with wild-type male dogs by natural mating or by artificial insemination. The bitches all became pregnant and successfully delivered 13 puppies (F1), of which two females were bred with wild-type dogs to deliver 7 offspring (F2), including 1 stillbirth. Among the 19 live offspring, 10 puppies showed emission of RFP under UV light and the presence of the RFP transgene was confirmed by genomic PCR and Southern blot analyses. In conclusion, transgenic RFP female dogs exhibited normal reproductive ability and expression of the transgene was demonstrated in F1 and F2 generations.

BACKGROUND Acute humoral xenograft rejection (AHXR) is an important barrier to xenograft survival. Human tumor necrosis factor-α (hTNF-α) is one of the essential mediators of AHXR and induces activation of porcine endothelial cells (PECs), resulting in upregulation of major histocompatibility complex molecules, adhesion molecules, and proinflammatory chemokines. We investigated whether introduction of a soluble human tumor necrosis factor receptor I-Fc (shTNFRI-Fc) fusion gene can suppress activation of PECs and, more importantly, produced shTNFRI-Fc transgenic pigs. METHODS The shTNFRI-Fc gene expression vector was constructed and inserted into PECs. The inhibitory effects of shTNFRI-Fc were tested by luciferase assay, reverse-transcriptase polymerase chain reaction, and flow cytometry. A shTNFRI-Fc transgenic pig was generated by somatic cell nuclear transfer. The expression of shTNFRI-Fc in the transgenic pig was evaluated by PCR, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. The inhibitory effects of shTNFRI-Fc in the serum obtained from the transgenic pig were also tested. RESULTS In comparison with control green fluorescent protein, shTNFRI-Fc protein showed much stronger inhibitory effects on NF-κB activation in the HEK293-NF-κB-luciferase reporting cell line, expression of chemokines and adhesion molecules in PECs, and TNF-α-mediated cytotoxicity. We successfully generated shTNFRI-Fc transgenic pig. Sera obtained from the transgenic pig inhibited induction of chemokines, and E-selectin in PECs stimulated with Human TNF-α. CONCLUSIONS We have generated transgenic pigs producing shTNFRI-Fc protein that can inhibit TNF-α-mediated activation of PECs. Because TNF-α is an important mediator of xenograft rejection, the use of xenografts that can produce shTNFRI-Fc proteins de novo could be an effective approach in overcoming a considerable component of the xenograft rejection process, especially AHXR.

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.

SummaryIn non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus–oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5–5.5% CO2 under various conditions (37–39 °C and 5–20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.

Genetically manipulated transparent animals were already explored in many species for in vivo study of gene expression, transplantation analysis and cancer biology. However, there are no reports about transparent animals as in vitro genetic resources. In the present study, fin-derived cells from glass catfish (Krytopterus bicirrhis), naturally transparent fish with a visible skeleton and internal organs, were isolated after culturing fin explants and characterized using cryopreservation and cell cycle analysis. The cells grew well in DMEM (Dulbecco's modified Eagle's medium) containing 1% (v/v) P/S (penicillin-streptomycin) and 10% (v/v) fetal bovine serum at 26°C and showed increased cryopreservation efficiency with the slow-freezing method in the presence of 15% dimethyl sulfoxide. In addition, cell cycle analysis was evaluated based on flow cytometric analysis, and culturing to confluence (85%) was more effective for synchronizing cells at the G(0)/G(1) stages than roscovitine treatment (75%). This is the first report about cell isolation from transparent animals. The results from testing the cell's viability following cryopreservation and subjecting the cells to cycle analysis can be useful tools for genetic resource management.

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

Synchronization of the donor cell cycle is an important factor for successful animal cloning by nuclear transfer. To improve the efficiency of porcine cloning, in the present report, we evaluated effects of contact inhibition, serum starvation and roscovitine treatment of donor cells on in vitro and in vivo developmental potency of cloned porcine embryos. Fibroblasts derived from a porcine foetus at day 30 of gestation were isolated and cultured to 70% confluency. Then, cells were either cultured to 100% confluency for contact inhibition, or cultured in 0.5% serum for 72 h for serum starvation or with 15 μM roscovitine for 24 h. Cells were most effectively synchronized at G0/G1 in the serum starvation group (87.5%) compared with the contact inhibition and roscovitine treatment groups (76.3% and 79.9% respectively p < 0.05). However, after somatic cell nuclear transfer followed by in vitro culture, the serum starvation group showed a significantly lower blastocyst formation rate (5.6%) compared with the contact inhibition and roscovitine treatment groups (11.6% and 20.0% respectively). Differential expression of apoptosis-related genes and the level of apoptosis in each treatment group explain the variation in developmental competence among the groups. Significantly higher level of apoptosis was observed in the serum starvation group. On the other hand, the roscovitine treatment group shows the lowest level of apoptosis and the best in vitro development among the groups. Cloned embryos derived from roscovitine-treated donor cells were transferred to surrogate pigs. Three healthy live piglets were produced. In conclusion, we suggest that roscovitine treatment of donor cells improves development of cloned porcine embryos and can raise the efficiency of cloned piglet production.

We have established a new 4 stage epiblast isolation method from whole bovine cloned blastocysts without using immunosurgery. The new "peeling" method consists of dissolution of the zona pellucida (first stage), elimination of mural trophoblast (second stage), isolation of primitive endoderm and epiblast from polar trophoblast (third stage), and isolation of epiblast from primitive endoderm (fourth stage). The bovine cloned blastocyst consists of 4 different types of cells showing abundant alkaline phosphatase activity. The epiblast origin of isolated cells was confirmed by in vitro differentiation of isolated cells to tubulin beta3-positive neurons and by embryoid body formation. The bovine cloned blastocyst origin of isolated epiblasts was confirmed by microsatellite analysis and mitochondrial DNA sequencing analysis. This new method might accelerate establishment of somatic cell nuclear transfer derived embryonic stem cell lines from bovine and other mammals.

SummaryCycle synchronization of donor cells in the G0/G1stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents – dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) – on synchronization of canine fibroblasts at the G0/G1stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1(77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo development. The present study evaluated temporal effects of alpha-tocopherol and L-ascorbic acid on the porcine embryo development, and investigated whether a single or twice supplements of these two antioxidants at a divided concentrations favors the embryo development. In order to determine temporal effects of alpha-tocopherol and/or L-ascorbic acid, 100 microM alpha-tocopherol or 200 microM L-ascorbic acid were supplemented to the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In another set of experiments, the concentration was divided into two equal halves, i.e., 50 microM alpha-tocopherol and 100 microM L-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture. Supplementing culture media with 100 microM alpha-tocopherol for the entire culture period of 168 h or starting from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting from the 96 or 120 h of culture. L-Ascorbic acid (200 microM) alone or together with alpha-tocopherol (100 microM) with a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst. L-ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control. No synergistic effect was observed on embryo development at a single supplement of these antioxidants. Although, at divided supplements higher blastocyst percentage was observed compared with control group, no further beneficial effect was observed compared with alpha-tocopherol or L-ascorbic acid alone. Our results demonstrated that the embryotrophic effects of alpha-tocopherol and/or L-ascorbic acid, in terms of frequency of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation timing.