The Influence of Increasing Roughage Content in the Diet on the Growth Performance and Intestinal Flora of Jinwu and Duroc × Landrace × Yorkshire Pigs.

The Jinwu pig (JW) is a hybrid breed originating from the Chinese indigenous Jinhua pig and Duroc pig, boasting excellent meat quality and fast growth rates. This study aimed to verify the tolerance of JW to roughage, similar to most Chinese indigenous pigs. In this research, two types of feed were provided to JW and Duroc × Landrace × Yorkshire pigs (DLY): a basal diet and a roughage diet (increasing the rice bran and wheat bran content in the basal diet from 23% to 40%) for a 65-day experimental period. The roughage diet showed an increasing trend in the feed conversion ratio (F/G), with a 17.61% increase in feed consumption per unit weight gain for DLY, while the increase for JW was only 4.26%. A 16S rRNA sequencing analysis revealed that the roughage diet increased the relative abundance of beneficial bacteria, such as Lactobacillus and Clostridium, while reducing the relative abundance of some potential pathogens, thus improving the gut microbiota environment. After being fed with the roughage diet, the abundance of bacterial genera, such as Treponema, Terrisporobacter, Coprococcus, and Ruminococcaceae, which aid in the digestion and utilization of dietary fiber, were significantly higher in Jinwu compared to DLY, indicating that these bacterial genera confer Jinwu with a higher tolerance to roughage than DLY.

CRISPR/Cas9-meditated gene knockout in pigs proves that LGALS12 deficiency suppresses the proliferation and differentiation of porcine adipocytes.

LGALS12, also known as galectin12, belongs to the galectin family with ß-galactoside-binding activity. We previously reported that LGALS12 is an important regulator of adipogenesis in porcine adipocytes in vitro, but its value in pig breeding needed to be explored in vivo. In this study, we used CRISPR/Cas9 to construct porcine fetal fibroblasts (PFFs) with a 43 bp deletion in LGALS12 exon 2. Using these PFFs as donor cells, a LGALS12 knockout pig model was generated via somatic cell nuclear transfer. Primary cultures of porcine intramuscular (IM) and subcutaneous (SC) adipocytes were established using cells from LGALS12 knockout pigs and wild-type pigs. A comparison of these cells proved that LGALS12 deficiency suppresses cell proliferation via the RAS-p38MAPK pathway and promotes lipolysis via the PKA pathway in both IM and SC adipocytes. In addition, we observed AKT activation only in IM adipocytes and suppression of the Wnt/ß-catenin only in SC adipocytes. Our findings suggest that LGALS12 deficiency affects the adipogenesis of IM and SC adipocytes through different mechanisms.

The effect of fixed-time artificial insemination protocol initiated at different stages of the estrous cycle on follicle development and ovulation in gilts.

Hormonal products have been developed for fixed-time artificial insemination (FTAI) to improve the efficiency of swine production. Here, we evaluated the effect of an FTAI protocol initiated during different phases of the estrous cycle on follicle development and ovulation in gilts. A total of 36 gilts were equally divided into three groups designated as the luteal (L), follicular (F), and post-ovulation (O) groups and fed with 20 mg of altrenogest for 18 days, followed by intramuscular injection of 1000 IU PMSG at 42 h after withdrawal of altrenogest, and 100 µg of GnRH after an 80-h interval. The L group had the highest number of follicles 4-6 mm in diameter, as well as corpora hemorrhagica. The mRNA expression of caspase-9 in the L group were significantly lower than those in the O and F groups (P < 0.05), while CYP11A1 and VEGF mRNA expression levels were significantly higher (P < 0.05). Moreover, FSHR mRNA levels were significantly higher in the O group than in the L, F, and control groups (P < 0.05). LHCGR and CYP19A1 mRNA levels were the highest in the F group (P < 0.05). Thus, the changes in the expression of genes associated with follicular development, maturation, and ovulation identified in this study indicated that initiation of the FTAI protocol during the luteal phase induced a better environment for follicle development and ovulation in gilts.

Improving ovulation in gilts using anti-inhibin serum treatment combined with fixed-time artificial insemination.

For successful batch farrowing, porcine oestrus and ovulation must be synchronized using fixed-time artificial insemination (FTAI). However, exogenous gonadotropins, which are currently used in FTAI, negatively affect gilt ovulation. Here, we aimed to improve sexually mature gilt superovulation efficiency using passive immunization against inhibin during FTAI. Altrenogest-treated gilts were challenged with 10 ml anti-inhibin serum (AIS group, n = 6), 1,000 IU pregnant mare serum gonadotropin (PMSG group, n = 6), or 10 ml goat serum (control group, n = 6). Gilts in the AIS and PMSG groups were inseminated according to the FTAI protocol, and gilts in the control group were inseminated during natural oestrus. When PMSG was replaced by AIS during FTAI of gilts, ovulation rate and embryos recovered were significantly greater in the AIS group as compared to the other two groups (p < .05). Especially the average number of 6-8-cell embryos in the AIS group was significantly higher than that in the PMSG group (p < .01). Moreover, the blastocyst number in the AIS group was significantly higher than that in the PMSG group and the control group (p < .05). But there was no significant difference in the blastocyst number between the PMSG group and the control group (p > .05). Besides, plasma levels of estradiol-ß (E2) and progesterone (P4) were significantly greater in the AIS group as compared to the other two groups on Day 23 and D 27, respectively (p < .01). In summary, we devised an improved high-yield FTAI protocol for sexually mature gilts using AIS; this protocol had a greater superovulation efficiency than the FTAI using PMSG.

Isolation, characterization and germline chimera preparation of primordial germ cells from the Chinese Meiling chicken.

Primordial germ cells (PGCs) are precursors of germline cells that can generate sperm and eggs in adults, making them promising tools for transgenic animal preparation and germplasm preservation, especially in avians. In this study, we purified the PGCs from circulating embryonic blood of Chinese Meiling chickens using Nycodenz density centrifugation, and characterized them by alkaline phosphatase (AKP) staining, periodic acid-Schiff (PAS) staining and stage-specific embryonic antigen-1 (SSEA-1) immunostaining and PGC-specific gene amplification. The purified PGCs were also labeled with PKH26 and transferred into donor chicken embryos at the Hamburger-Hamilton (HH) stage 14 to 16, and cells with red fluorescence were observed in the gonads of 8-d-old embryos. When using about 200 PGCs isolated from Chinese Meiling chickens, microinjection into the dorsal aortas of recipient chickens with white feathers at stage HH14 to 16 resulted in germline chimeras that hatched and attained sexual maturity. The frequency of donor-derived yellow-feathered offspring from germline chimeric chickens was 12.6 ± 2.6% after mating with the white-feathered chickens. These results demonstrate that we had successfully purified the PGCs from the Chinese Meiling chicken. These germline cells could be used to preserve Chinese Meiling chickens.

Effect of shRNA-mediated Xist knockdown on the quality of porcine parthenogenetic embryos.

BACKGROUND: Parthenogenetically activated oocytes exhibit poor embryo development and lower total numbers of cells per blastocyst accompanied by abnormally increased expression of Xist, a long noncoding RNA that plays an important role in triggering X chromosome inactivation during embryogenesis. RESULTS: To investigate whether knockdown of Xist influences parthenogenetic development in pigs. We developed an anti-Xist short hairpin RNA (shRNA) vector, which can significantly inhibit Xist expression for at least seven days when injected at 12-13 hr after parthenogenetic activation. Embryonic cleavage, blastocyst formation, and total blastocyst cell numbers were compared during the blastocyst stage, as well as the expression of an X-linked gene and three pluripotent transcription factors. Knockdown of Xist significantly increases the total blastocyst cell number, but does not influence the rate of embryo cleavage and blastocyst formation. The expressions of Sox2, Nanog, and Oct4 were also significantly improved in the injected embryos compared with the control at the blastocyst stage, but the Foxp3 expression level was not increased significantly. CONCLUSIONS: The present study provides valuable information for understanding the role of Xist in parthenogenesis and presents a new approach for improving the quality of porcine parthenogenetic embryos. Developmental Dynamics 248:140-148, 2019. © 2018 Wiley Periodicals, Inc.

Jun dimerization protein 2 controls hypoxia-induced replicative senescence via both the p16Ink4a-pRb and Arf-p53 pathways.

The main regulators of replicative senescence in mice are p16Ink4a and Arf, inhibitors of cell cycle progression. Jun dimerization protein 2 (JDP2)-deficient mouse embryonic fibroblasts are resistant to replicative senescence through recruitment of the Polycomb repressive complexes 1 and 2 to the promoter of the gene that encodes p16Ink4a and inhibits the methylation of lysine 27 of the histone H3 locus. However, whether or not JDP2 is able to regulate the chromatin signaling of either p16Ink4a-pRb or Arf-p53, or both, in response to oxidative stress remains elusive. Thus, this study sought to clarify this point. We demonstrated that the introduction of JDP2 leads to upregulation of p16Ink4a and Arf and decreases cell proliferation in the presence of environmental (20% O2), but not in low (3% O2) oxygen. JDP2-mediated growth suppression was inhibited by the downregulation of both p16Ink4a and Arf. Conversely, the forced expression of p16Ink4a or Arf inhibited cell growth even in the absence of JDP2. The downregulation of both the p53 and pRb pathways, but not each individually, was sufficient to block JDP2-dependent growth inhibition. These data suggest that JDP2 induces p16Ink4a and Arf by mediating signals from oxidative stress, resulting in cell cycle arrest via both the p16Ink4a-pRb and Arf-p53 pathways.

Production of germline transgenic pigs co-expressing double fluorescent proteins by lentiviral vector.

Genomic integration of transgene by lentiviral vector has been proved an efficient method to produce single-transgenic animals. But it failed to create multi-gene transgenic offspring. Here, we have exploited lentivirus to generate the double-transgenic piglets through the female germline. The recombinant lentivirus containing fluorescent proteins genes (DsRed1 and Venus) were injected into the perivitelline space of 2-cell stage in vitro porcine embryos. Compared to control group, there was no significantly decreased in the proportion of blastocysts, and the two fluorescent protein genes were co-expressed in almost all the injected embryos. Total of 32 injected in vitro embryos were transferred to 2 recipients. One recipient gave birth of three live offspring, and one female piglet was identified as genomic transgene integration by PCR analysis. Subsequently, the female transgenic founder was mated naturally with a wild-type boar and gave birth of two litters of total 23 F(1) generation piglets, among which Venus and DsRed1 genes were detected in 11 piglets and 10 kinds of organs by PCR and RT-PCR respectively. The co-expression of two fluorescent proteins was visible in four different frozen tissue sections from the RT-PCR positive piglets, and 3 to 5 copies of the transgenes were detected to be integrated into the second generation genome by southern blotting analysis. The transgenes were heritable and stably integrated in the F(1) generation. The results indicated for the first time that lentiviral vector combined with natural mating has the potential to become a simple and practical technology to create germline double-transgenic livestock or biomedical animals.

An easy method for preparation of Cre-loxP regulated fluorescent adenoviral expression vectors and its application for direct reprogramming into hepatocytes.

The recombinant adenoviral gene expression system is a powerful tool for gene delivery. However, it is difficult to obtain high titers of infectious virus, principally due to the toxicity of the expressed gene which affects on virus replication in the host HEK293 cells. To avoid these problems, we generated a Cre-loxP-regulated fluorescent universal vector (termed pAxCALRL). This vector produces recombinant adenoviruses that express the red fluorescent protein (RFP) instead of the inserted gene during proliferation, which limits toxicity and can be used to monitor viral replication. Expression of the gene of interest is induced by co-infection with an adenovirus that expresses Cre-recombinase (AxCANCre). Recombinant adenovirus produced by this system that express Hnf4α and Foxa2 were used to reprogram mouse embryo fibroblast (MEF) into induced-hepatocyte-like cells (iHep) following several rounds of infection, demonstrating the efficacy of this new system.

Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters.

The promoter plays an important role in the regulation of gene expression. To analyze a promoter's activity, we developed a novel lentiviral T/A vector that contains two reporter genes, a luciferase (Luc2) gene and a green fluorescent protein (Venus) gene, that are linked via an internal ribosome entry site (IRES2). To test the performance of this vector, phosphoglycerate kinase-1 (PGK) and elongation factor-1α (EF1α) promoters were amplified by PCR and inserted into this lentiviral T/A vector using T4 DNA ligase, yielding two promoter-reporter vectors: pLent-T-PGK and pLent-T-EF1α. When these vectors were transfected into 293T cells, we observed a higher level of Venus expression under a fluorescence microscopy in the case of pLent-T-EF1α as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1α and PGK was approximately 9:1. The two promoter-reporter vectors were also packaged as lentiviral particles to conduct promoter activity assay in cultured cells. The ratio of the promoter activities of EF1α and PGK was 4.23:1 when they were infected into 293T cells at a multiplicity of infection of 1. This value is comparable to that of a parallel experiment using the commercial luciferase reporter vector pGL4.10 with an activity ratio of 5.99:1 for EF1α and PGK. These results indicate that lentiviral T/A vector will be a useful tool for analysis of promoter activity and specificity.

Cloning and characterization of the mouse JDP2 gene promoter reveal negative regulation by p53.

Jun dimerization protein 2 (JDP2) is a repressor of transcription factor AP-1. To investigate the transcriptional regulation of the JDP2 gene, we cloned the 5'-flanking region of the mouse JDP2 gene. Primer extension analysis revealed a new transcription start site (+1). Promoter analysis showed that the region from nt -343 to nt +177 contains basal transcriptional activity. Interestingly, the tumor suppressor p53 significantly repressed the transcriptional activity of the JDP2 promoter. Given that JDP2 inhibits expression of p53, our results suggest a negative feedback loop between JDP2 and p53, and a direct link between JDP2 and a key oncogenic pathway.

Molecular characterization of the porcine STAT4 and STAT6 genes.

Signal transducers and activators of transcription (STATs) are members of a recently identified family of transcription factors that activate gene transcription in response to a number of different cytokines. STAT4 and STAT6 were activated by interleukin (IL)-12 and IL-4 stimulation, which were important for the generation of Th1 and Th2 cells. In this study, we cloned the cDNA sequences and analyzed the genomic structure of porcine STAT4 (poSTAT4) and STAT6 (poSTAT6) genes. Chromosome localization assigned these two genes to SSC15 and SSC5, and they were most closely linked to maker SWR1002 and DK. The RT-PCR revealed that both genes were expressed in eight diverse tissues, with the highest level in small intestine, followed by lung, kidney, muscle and stomach, whereas expressions in heart, liver and spleen were relatively weak. Transient transfection indicated that poSTAT4 and poSTAT6 proteins distributed throughout the whole porcine hip artery endothelial cell. A single nucleotide polymorphism (A/G), which can be recognized by restriction enzyme TaiI, was identified at the 3' untranslated region of poSTAT6, and genotyping results showed apparent variation in allele frequency between Chinese indigenous and western breeds.

Genetic materials at the gene engineering division, RIKEN BioResource Center.

Genetic materials are one of the most important and fundamental research resources for studying biological phenomena. Scientific need for genetic materials has been increasing and will never cease. Ever since it was established as RIKEN DNA Bank in 1987, the Gene Engineering Division of RIKEN BioResource Center (BRC) has been engaged in the collection, maintenance, storage, propagation, quality control, and distribution of genetic resources developed mainly by the Japanese research community. When RIKEN BRC was inaugurated in 2001, RIKEN DNA Bank was incorporated as one of its six Divisions, the Gene Engineering Division. The Gene Engineering Division was selected as a core facility for the genetic resources of mammalian and microbe origin by the National BioResource Project (NBRP) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan in 2002. With support from the scientific community, the Division now holds over 3 million clones of genetic materials for distribution. The genetic resources include cloned DNAs, gene libraries (e.g., cDNA and genomic DNA cloned into phage, cosmid, BAC, phosmid, and YAC), vectors, hosts, recombinant viruses, and ordered library sets derived from animal cells, including human and mouse cells, microorganisms, and viruses. Recently genetic materials produced by a few MEXT national research projects were transferred to the Gene Engineering Division for further dissemination. The Gene Engineering Division performs rigorous quality control of reproducibility, restriction enzyme mapping and nucleotide sequences of clones to ensure the reproducibility of in vivo and in vitro experiments. Users can easily access our genetic materials through the internet and obtain the DNA resources for a minimal fee. Not only the materials, but also information of features and technology related to the materials are provided via the web site of RIKEN BRC. Training courses are also given to transfer the technology for handling viral vectors. RIKEN BRC supports scientists around the world in the use of valuable genetic materials.

JDP2 (Jun Dimerization Protein 2)-deficient mouse embryonic fibroblasts are resistant to replicative senescence.

JDP2 (Jun dimerization protein 2, an AP-1 transcription factor) is involved in the regulation of the differentiation and proliferation of cells. We report here that JDP2-deficient mouse embryonic fibroblasts (Jdp2(-/-) MEF) are resistant to replicative senescence. In the absence of JDP2, the level of expression of p16(Ink4a), which is known to rise as normal fibroblasts age, fell significantly when cells were cultured for more than 2 months. Conversely, the overexpression of JDP2 induced the expression of genes for p16(Ink4a) and p19(Arf). Moreover, at the promoter of the gene for p16(Ink4a) in Jdp2(-/-) MEF, the extent of methylation of lysine 27 of histone H3 (H3K27), which is important for gene silencing, increased. Polycomb-repressive complexes (PRC-1 and PRC-2), which are responsible for histone methylation, bound efficiently to the promoter to repress the expression of the gene for p16(Ink4a). As a result, JDP2-deficient MEF became resistant to replicative senescence. Our results indicate that JDP2 is involved in the signaling pathway for senescence via epigenetic regulation of the expression of the gene for p16(Ink4a).

The ubiquitously expressed bZIP inhibitor, JDP2, suppresses the transcription of its homologue immediate early gene counterpart, ATF3.

JDP2 is a ubiquitously expressed bZIP repressor protein. JDP2 binds TPA response element and cyclic AMP response element located within various promoters. JDP2 displays a high degree of homology to the immediate early gene ATF3. ATF3 plays a crucial role in the cellular adaptive response to multiple stress insults as well as growth stimuli. We have identified ATF3 as a potential target gene for JDP2 repression. JDP2 regulates the ATF3 promoter potentially through binding to both the consensus ATF/CRE site and a non-consensus ATF3 auto-repression DNA-binding element. Expression of ATF3 protein in wild-type mouse embryo fibroblast (MEF) cells is below the detectable levels, whereas, JDP2 disrupted MEF cells display noticeable level of ATF3 protein. Following either serum or ER stress stimulation, ATF3 expression is potentiated in JDP2-KO fibroblast cells as compared with wild-type cells. Mice with either JDP2 over-expression or JDP2 disruption display undetectable level of ATF3 protein. However, ATF3 induction in response to either growth or stress signals is dependent on JDP2 expression level. ATF3 induction is attenuated in JDP2 over-expressing mice whereas is potentiated in JDP2-KO mice as compared with the corresponding wild-type mice. Collectively, the data presented strongly suggest that JDP2 plays a role in the determination of the ATF3 adaptive cellular threshold response to different stress insults and growth stimuli.

Phosphorylation of Activation Transcription Factor-2 at Serine 121 by Protein Kinase C Controls c-Jun-mediated Activation of Transcription.

Activation transcription factor-2 (ATF-2) is phosphorylated by various protein kinases, such as JNK/p38/ERK, calmodulin kinase IV, protein kinase A, and protein kinase C (PKC), in response to a variety of stimuli. However, the role of the phosphorylation of ATF-2 by PKC in vivo in the transcriptional control of genes that include the activation protein-1 (AP-1)/cyclic AMP-response element remains to be defined. Using antibodies against the phosphorylated serine residue (Ser(P)) at position 121 of ATF-2, we have demonstrated that PKC phosphorylates ATF-2 at Ser-121 and that phosphorylation of Ser-121 (to yield ATF-2pS121) becomes detectable at the late stage of the response of HeLa cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) and is maintained for more than 2 h. By contrast, phosphorylation of ATF-2 at threonine residues 69 and 71 (Thr-69/71, to yield ATF-2pT69/71) and at Ser-340 and Ser-367 (to yield ATF-2pS340 and ATF-2pS367) is detectable as an immediate early response. Unlike levels of ATF-2pT69/71 and ATF-2pS340, the level of ATF-2pS121 increases in the nuclei of HeLa cells in response to TPA. A serine-to-alanine mutation at position 121 of ATF-2 represses the c-Jun-dependent transcription of AP-1/cyclic AMP-response element reporter genes and also the p300-mediated activation of a Gal4-reporter gene in response to TPA. Our results suggest that the phosphorylation of ATF-2 at Ser-121 plays a key role in the c-Jun-mediated activation of transcription that occurs in response to TPA.

Regulation of histone acetylation and nucleosome assembly by transcription factor JDP2.

Jun dimerization protein-2 (JDP2) is a component of the AP-1 transcription factor that represses transactivation mediated by the Jun family of proteins. Here, we examine the functional mechanisms of JDP2 and show that it can inhibit p300-mediated acetylation of core histones in vitro and in vivo. Inhibition of histone acetylation requires the N-terminal 35 residues and the DNA-binding region of JDP2. In addition, we demonstrate that JDP2 has histone-chaperone activity in vitro. These results suggest that the sequence-specific DNA-binding protein JDP2 may control transcription via direct regulation of the modification of histones and the assembly of chromatin.

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank.

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration.

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35x10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.