Improving the Efficiency of CRISPR Ribonucleoprotein-Mediated Precise Gene Editing by Small Molecules in Porcine Fibroblasts.

The aim of this study was to verify whether small molecules can improve the efficiency of precision gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoprotein (RNP) in porcine cells. CRISPR associated 9 (Cas9) protein, small guide RNA (sgRNA), phosphorothioate-modified single-stranded oligonucleotides (ssODN), and different small molecules were used to generate precise nucleotide substitutions at the insulin (INS) gene by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by polymerase chain reaction (PCR) for the target site. All samples were sequenced and analyzed, and the efficiencies of different small molecules at the target site were compared. The results showed that the optimal concentrations of the small molecules, including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A, for in vitro-cultured PFFs' viability were determined. Compared with the control group, the single small molecules including L-189, NU7441, SCR7, L755507, RS-1, and Brefeldin A increased the efficiency of HDR-mediated precise gene editing from 1.71-fold to 2.28-fold, respectively. There are no benefits in using the combination of two small molecules, since none of the combinations improved the precise gene editing efficiency compared to single small molecules. In conclusion, these results suggested that a single small molecule can increase the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells.

CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts.

The multi-gene editing porcine cell model can analyze the genetic mechanisms of multiple genes, which is beneficial for accelerating genetic breeding. However, there has been a lack of an effective strategy to simultaneously perform precise multi-gene editing in porcine cells. In this study, we aimed to improve the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells. CRISPR RNP, including Cas9 protein, sgRNA, and ssODN, was used to generate precise nucleotide substitutions by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by PCR for each target site. To enhance HDR efficacy, small-molecule M3814 and phosphorothioate-modified ssODN were employed. All target DNA samples were sequenced and analyzed, and the efficiencies of different combinations of the CRISPR RNP system in target sites were compared. The results showed that when 2 µM M3814, a small molecule which inhibits NHEJ-mediated repair by blocking DNA-PKs activity, was used, there was no toxicity to PFFs. The CRISPR RNP-mediated HDR efficiency increased 3.62-fold. The combination of CRISPR RNP with 2 µM M3814 and PS-ssODNs achieved an HDR-mediated precision gene modification efficiency of approximately 42.81% in mutated cells, a 6.38-fold increase compared to the control group. Then, we used the optimized CRISPR RNP system to perform simultaneous editing of two and three loci at the INS and RLN3 genes. The results showed that the CRISPR RNP system could simultaneously edit two and three loci. The efficiency of simultaneous editing of two loci was not significantly different from that of single-gene editing compared to the efficiency of single-locus editing. The efficiency of simultaneous precise editing of INS, RLN3 exon 1, and RLN3 exon 2 was 0.29%, 0.24%, and 1.05%, respectively. This study demonstrated that a 2 µM M3814 combination with PS-ssODNs improves the efficacy of CRISPR RNP-mediated precise gene editing and allows for precise editing of up to three genes simultaneously in porcine cells.

Single-Cell RNA Sequencing Reveals Differences in Chromatin Remodeling and Energy Metabolism among In Vivo-Developed, In Vitro-Fertilized, and Parthenogenetically Activated Embryos from the Oocyte to 8-Cell Stages in Pigs.

In vitro-fertilized (IVF) and parthenogenetically activated (PA) embryos, key to genetic engineering, face more developmental challenges than in vivo-developed embryos (IVV). We analyzed single-cell RNA-seq data from the oocyte to eight-cell stages in IVV, IVF, and PA porcine embryos, focusing on developmental differences during early zygotic genome activation (ZGA), a vital stage for embryonic development. (1) Our findings reveal that in vitro embryos (IVF and PA) exhibit more similar developmental trajectories compared to IVV embryos, with PA embryos showing the least gene diversity at each stage. (2) Significant differences in maternal mRNA, particularly affecting mRNA splicing, energy metabolism, and chromatin remodeling, were observed. Key genes like SMARCB1 (in vivo) and SIRT1 (in vitro) played major roles, with HDAC1 (in vivo) and EZH2 (in vitro) likely central in their complexes. (3) Across different types of embryos, there was minimal overlap in gene upregulation during ZGA, with IVV embryos demonstrating more pronounced upregulation. During minor ZGA, global epigenetic modification patterns diverged and expanded further. Specifically, in IVV, genes, especially those linked to H4 acetylation and H2 ubiquitination, were more actively regulated compared to PA embryos, which showed an increase in H3 methylation. Additionally, both types displayed a distinction in DNA methylation. During major ZGA, IVV distinctively upregulated genes related to mitochondrial regulation, ATP synthesis, and oxidative phosphorylation. (4) Furthermore, disparities in mRNA degradation-related genes between in vivo and in vitro embryos were more pronounced during major ZGA. In IVV, there was significant maternal mRNA degradation. Maternal genes regulating phosphatase activity and cell junctions, highly expressed in both in vivo and in vitro embryos, were degraded in IVV in a timely manner but not in in vitro embryos. (5) Our analysis also highlighted a higher expression of many mitochondrially encoded genes in in vitro embryos, yet their nucleosome occupancy and the ATP8 expression were notably higher in IVV.

Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript.

Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem-loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications.

Multiplexed genome engineering for porcine fetal fibroblasts with gRNA-tRNA arrays based on CRISPR/Cas9.

Multiplex gene modifications are highly required for various fields of porcine research. In many species, the CRISPR/Cas9 system has been widely applied for genomic editing and provides a potential tool for introducing multiplex genome mutations simultaneously. Here, we present a CRISPR-Cas9 gRNA-tRNA array (GTR-CRISPR) for multiplexed engineering of porcine fetal fibroblasts (PFFs). We successfully produced multiple sgRNAs using only one Pol III promoter by taking advantage of the endogenous tRNA processing mechanism in porcine cells. Using an all-in-one construct carrying GTR and Cas9, we disrupted the IGFBP3, MSTN, MC4R, and SOCS2 genes in multiple codon regions in one PFF cell simultaneously. This technique allows the simultaneous disruption of four genes with 5.5% efficiency. As a result, this approach may effectively target multiple genes at the same time, making it a powerful tool for establishing multiple genes mutant cells in pigs.

Exploring differentially expressed genes related to metabolism by RNA-Seq in porcine embryonic fibroblast after insulin treatment.

BACKGROUND: Insulin regulates glucose homeostasis and has important effects on metabolism, cell growth, and differentiation. Depending on the cell type and physiological context, insulin signal has specific pathways and biological outcomes in different tissues and cells. For studying the signal pathway of insulin on glycolipid metabolism in porcine embryonic fibroblast (PEF), we used high-throughput sequencing to monitor gene expression patterns regulated by insulin. OBJECTIVES: The goal of our research was to see how insulin affected glucose and lipid metabolism in PEFs. METHODS: We cultured the PEFs with the addition of insulin and sampled them at 0, 48, and 72 h for RNA-Seq analysis in triplicate for each time point. RESULTS: At 48 and 72 h, 801 and 1,176 genes were differentially expressed, respectively. Of these, 272 up-regulated genes and 264 down-regulated genes were common to both time points. Gene Ontology analysis was used to annotate the functions of the differentially expressed genes (DEGs), the biological processes related to lipid metabolism and cell cycle were dominant. And the DEGs were significantly enriched in interleukin-17 signaling pathway, phosphatidylinositol-3-kinase-protein kinase B signaling pathway, pyruvate metabolism, and others pathways related to lipid metabolism by Kyoto Encyclopedia of Genes and Genomes enrichment analysis. CONCLUSIONS: These results elucidate the transcriptomic response to insulin in PEF. The genes and pathways involved in the transcriptome mechanisms provide useful information for further research into the complicated molecular processes of insulin in PEF.

Atorvastatin suppresses lipopolysaccharide-induced inflammation in human coronary artery endothelial cells

Abstract The present study was designed to examine the effects of atorvastatin on vascular inflammatory responses in human coronary artery endothelial cells(HCAECs), when challenged by lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR4) ligand. HCAECs were pretreated with atorvastatin and induced by LPS. The expression of TLR4, interleukin -6(IL-6), monocyte chemoattractant protein 1(MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecular-1(ICAM-1), nuclear factor-κB (NF-κB) and p38 mitogen activated protein kinase(p38 MAPK) were evaluated using Real-time polymerase chain reaction, cytokine ELISA assay and Western blotting. The results showed that pretreatment with atorvastatin down-regulated the expression of TLR4 in LPS-activated HCAECs. Atorvastatin also attenuated the LPS-induced expression of interleukin IL-6 and MCP-1, at both the transcription and translation level in HCAECs. LPS-induced endothelial cell adhesion molecules, ICAM-1 and VCAM-1 expression were also reduced by pretreatment with atorvastatin. Furthermore, atorvastatin efficiently suppressed LPS-induced phosphorylation of NF-κB and p38 MAPK in HCAECs. These findings show that atorvastatin suppresses endothelial cell inflammation, suggesting that atorvastatin may be suitable for development as a therapeutic agent for inflammatory cardiovascular disease.

Argonaute 2 is a key regulator of maternal mRNA degradation in mouse early embryos.

In mammalian early embryos, the transition from maternal to embryonic control of gene expression requires timely degradation of a subset of maternal mRNAs (MRD). Recently, zygotic genome activation (ZGA)-dependent MRD has been characterized in mouse 2-cell embryo. However, in early embryos, the dynamics of MRD is still poorly understood, and the maternal factor-mediated MRD before and along with ZGA has not been investigated. Argonaute 2 (Ago2) is highly expressed in mouse oocyte and early embryos. In this study, we showed that Ago2-dependent degradation involving RNA interference (RNAi) and RNA activation (RNAa) pathways contributes to the decay of over half of the maternal mRNAs in mouse early embryos. We demonstrated that AGO2 guided by endogenous small interfering RNAs (endosiRNAs), generated from double-stranded RNAs (dsRNAs) formed by maternal mRNAs with their complementary long noncoding RNAs (CMR-lncRNAs), could target maternal mRNAs and cooperate with P-bodies to promote MRD. In addition, we also showed that AGO2 may interact with small activating RNAs (saRNAs) to activate Yap1 and Tead4, triggering ZGA-dependent MRD. Thus, Ago2-dependent degradation is required for timely elimination of subgroups of maternal mRNAs and facilitates the transition between developmental states.

Cellular reprogramming by single-cell fusion with mouse embryonic stem cells in pig.

Fusion of differentiated somatic cells with pluripotent stem cells can be used for cellular reprogramming, but the efficiency to obtain hybrid cells is extremely low. Here, we explored a novel cell fusion system, termed single-cell fusion, the efficiency was significantly improved verified by fusion of mouse embryonic stem cells (mESCs), comparing to traditional polyethylene glycol fusion. Then, we employed the optimized system to perform cell fusion of porcine embryonic fibroblasts (PEFs) and porcine pluripotent stem cells (pPSCs) with mESCs. The hybrid cells showed both red and green fluorescence and expressed species-specific genes of mouse and pig to evidence that the fusion was successful. The hybrid cells displayed characteristics similar with mESCs, including colony morphology, alkaline phosphatase positive and formation of embryoid body, and the expressions of core pluripotent factors OCT4, NANOG, and SOX2 of the pig were induced in the mESC/PEF hybrid cells. The results indicate PEFs and pPSCs could be reprogrammed by mESCs via the single-cell fusion. Taking advantage of the hybrid cells to investigate the signaling pathways depended on the pluripotency of pig, we suggest the transforming growth factor-ß signaling pathways may play important roles. In summary, the single-cell fusion is highly efficient, and we believe in the future it will be widely used in the application and fundamental research.

The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells.

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.

Endo-siRNAs repress expression of SINE1B during in vitro maturation of porcine oocyte.

Approximately 40% of mammalian genome is made of transposable elements (TEs), and during specific biological processes, such as gametogenesis, they may be activated by global demethylation, so strict silencing mechanism is indispensable for genomic stability. Here, we performed small RNA-seq on Dicer1 knockdown (KD) oocytes in pig, and observed short interspersed nuclear elements 1B (SINE1B) derived endogenous small interfering RNAs (endo-siRNAs), termed SINE1B-siRNAs, were significantly decreased and their biogenesis was dependent on Dicer1 and transcript of SINE1B. Furthermore, by injection of mimics and inhibitors of the SINE1B-siRNAs into germinal vesicle-stage (GV-stage) oocytes, we found the maturation rate was significantly decreased by SINE1B-siRNAs, indicating the SINE1B-siRNAs are indispensible for in vitro maturation (IVM) of porcine oocyte. To figure out the mechanism, we checked the expression pattern and DNA methylation status of SINE1B during IVM of porcine oocytes, and demonstrated the SINE1B-siRNAs could repress SINE1B expression induced by hypomethylation at a post-transcriptional level. Our results suggest that during gametogenesis when the erasure of DNA methylation occurs, endo-siRNAs act as a chronic response to limit retrotransposon activation.

Endo-siRNAs regulate early embryonic development by inhibiting transcription of long terminal repeat sequence in pig†.

Activity of some endogenous retroviruses (ERVs) has been proven to be important for development of early mammalian embryo. However, abnormal activation of ERVs can also cause genetic diseases due to their ability to retrotranspose, so the regulatory mechanism to limit transcription of ERVs needs to be clarified. Endogenous small interfering RNA (endo-siRNA) has been reported to protect cells against transposable elements (TEs). Here, we determined the role of ERVs long terminal repeat sequences (LTRs) derived endo-siRNAs (LTR-siRNAs) on inhibition of the activity of ERVs during early embryonic development in pig. Seven most highly expressed LTR-siRNAs were identified in porcine zygote by high-throughput small RNA sequencing. We verified that the biogenesis of the LTR-siRNAs was DICER-dependent and they were generated from double-stranded RNA (dsRNA) formed by sense and antisense transcripts of LTRs. And, the expression of sense and antisense of LTRs might be due to the loss of DNA methylation at some LTR loci. Furthermore, we showed that the LTR-siRNAs could regulate early embryonic development by repression of LTRs expression at a post-transcriptional level. So, we propose here, during early embryonic development when epigenetic reprogramming occurs, the endo-siRNA pathway acts as a sophisticated balance of regulatory mechanism for ERV activity.

Effect of IGFBP2 Overexpression on the Expression of Fatty Acid Synthesis Genes in Primary Cultured Chicken Hepatocytes.

The effects of insulin-like growth factor binding protein 2 (IGFBP2) on the expression of fatty acid synthesis regulators and triglyceride production were investigated in primary cultured chicken hepatocytes. The full-length chicken IGFBP2 coding region was synthesized by overlap extension PCR and cloned into the pcDNA3.1 vector. An in situ digestion method was used to prepare the chicken hepatocytes. Primary chicken hepatocytes were maintained in monolayer culture. Real-time PCR was used to detect changes in the expression of IGFBP2, PPARG, IGF1, IGF1R, APOAI, and LFABP, after the overexpression of IGFBP2 in chicken hepatocytes. Triglyceride production and glucose content were also evaluated using triglyceride and glucose analysis methods. The expression level of IGFBP2 increased after transfection of the IGFBP2-containing vector. The expression levels of PPARG, IGF1, and IGF1R also increased in cultured chicken hepatocytes after the overexpression of IGFBP2, whereas the expression of LFABP and APOAI decreased. Triglyceride production in primary cultured chicken hepatocytes increased after the overexpression of IGFBP2. These results suggest that IGFBP2 is involved in lipogenesis, increasing both the expression of fatty acid synthesis regulators, and triglyceride production in primary cultured chicken hepatocytes.

Production of transgenic broilers by non-viral vectors via optimizing egg windowing and screening transgenic roosters.

The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.

Fetal bovine serum promotes the development of in vitro porcine blastocysts by activating the Rho-associated kinase signalling pathway.

Fetal bovine serum (FBS) supplementation has beneficial effects on invitro porcine embryonic development, but the underlying mechanisms are unclear. In the present study we found that the addition of FBS to PZM-3 increased the number of cells in porcine blastocysts and hatching rate invitro primarily by promoting proliferation of the inner cell mass and further differentiation. Moreover, based on the following results, we surmise that FBS benefits blastocyst development by activating Rho-associated kinase (ROCK) signalling: (1) the ROCK signalling inhibitor Y-27632 decreased the blastocyst rate and the number of cells in blastocysts, whereas FBS rescued the developmental failure induced by Y-27632; (2) the mRNA levels of two ROCK isoforms, ROCK1 and ROCK2, were significantly increased in blastocysts derived from medium containing FBS; and (3) FBS increased RhoA/Rho-kinase expression in the nucleus of embryonic cells. These results indicate that FBS promotes the invitro development of porcine embryos by activating ROCK signalling in a chemically defined medium.

Amyloid deposition in a mouse model humanized at the transthyretin and retinol-binding protein 4 loci.

Familial amyloidotic polyneuropathy is an autosomal dominant disorder caused by a point mutation in the transthyretin (TTR) gene. The process of TTR amyloidogenesis begins with rate-limiting dissociation of the TTR tetramer. Thus, the TTR stabilizers, such as Tafamidis and Diflunisal, are now in clinical trials. Mouse models will be useful to testing the efficacy of these drugs. Although several mouse models have been generated, they all express mouse Rbp4. Thus, human TTR associates with mouse RBP4, resulting in different kinetic and thermodynamic stability profiles of TTR tetramers. To overcome this problem, we previously produced humanized mouse strains at both the TTR and Rbp4 loci (Ttr hTTRVal30 , Ttr hTTRMet30 , and Rbp4 hRBP4 ). By mating these mice, we produced double-humanized mouse strains, Ttr hTTRVal30/hTTRVal30 :Rbp4 hRBP4/hRBP4 and Ttr hTTRVal30/Met30 :Rbp4 hRBP4/hRBP4 . We used conventional transgenic mouse strains on a wild-type (Ttr +/+ :Tg[6.0hTTRMet30]) or knockout Ttr background (Ttr-/-:Tg[6.0hTTRMet30]) as reference strains. The double-humanized mouse showed 1/25 of serum hTTR and 1/40 of serum hRBP4 levels. However, amyloid deposition was more pronounced in Ttr hTTRVal30/Met30 :Rbp4 hRBP4/hRBP4 than in conventional transgenic mouse strains. In addition, a similar amount of amyloid deposition was also observed in Ttr hTTRVal30/ hTTRVal30 :Rbp4 hRBP4/ hRBP4 mice that carried the wild-type human TTR gene. Furthermore, amyloid deposition was first observed in the sciatic nerve without any additional genetic change. In all strains, anti-TTR antibody-positive deposits were found in earlier age and at higher percentage than amyloid fibril deposition. In double-humanized mice, gel filtration analysis of serum revealed that most hTTR was free of hRBP4, suggesting importance of free TTR for amyloid deposition.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

CSP-1103 (CHF5074) stabilizes human transthyretin in healthy human subjects.

Hereditary amyloid polyneuropathy is a type of protein misfolding disease. Transthyretin (TTR) is a homotetrameric serum protein and TTR tetramer dissociation is the limiting step in amyloid fibril formation. Thus, prevention of TTR dissociation is a promising therapeutic approach and some TTR stabilizers have been approved for the treatment of TTR amyloidosis. CSP-1103 (CHF5074) is a non-steroidal anti-inflammatory derivative that lacks cyclooxygenase inhibitory activity. In vitro, CSP-1103 stabilizes the TTR tetramer by binding to the thyroxine (T4) binding site. We have previously shown that serum TTR levels were increased by oral CSP-1103 administration through stabilization of TTR tetramers in humanized mice at both the Ttr locus and the Rbp4 locus. To determine whether CSP-1103 stabilizes TTR tetramers in humans, multiple CSP-1103 oral doses were administered for two weeks to 48 healthy human volunteers in a double-blind, placebo-controlled, parallel-group study. CSP-1103 treatment stabilized TTR tetramers in a dose-dependent manner under normal or denaturing stress conditions, thereby increasing serum TTR levels. Preincubation of serum with CSP-1103 or diflunisal in vitro increased the TTR tetramer stability. Computer simulation analysis revealed that the binding affinities of CSP-1103 with TTR at pH 7.0 were similar to those of tafamidis, thus confirming that CSP-1103 has potent TTR-stabilizing activity.

Sequencing and analysis of the complete mitochondrial genome of Elaphe davidi (Squamata: Colubridae).

The mitochondrial genome sequence of Elaphe davidi is analyzed and presented for the first time in this work. The genome was 17,117 bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 2 control region. The overall base composition is A (35.4%), C (25.2%), T (27.0%), and G (12.4%). The base compositions present clearly the A-T skew, which was most obviously in the control region and protein-coding genes. Mitochondrial genomes analyses based on MP, ML, NJ and Bayesian analyses yielded identical phylogenetic trees, indicating a close phylogenetic affinity of the 12 Colubridae species. Two major phyletic lineages were present in Colubridae. A clade included the six species (Dinodon semicarinatus, E. poryphyracea, Oocatochus rufodorsatus, Orthriophis taeniurus, E. bimaculata and E. davidi) of subfamily Colubrinae except for Oligodon ningshaanensis. Another clade (Hypsiglena chlorophaea, H. unaocularus, H. torquata and Imantodes cenchoa) included Thermophis zhaoermii and O. ningshaanensis as the sister taxon to Colubrinae. The genus Elaphe, Dinodon, Oocatochus and Orthriophis formed a monophyletic group with the high bootstrap value (100 %) in all examinations.

Trichostatin A rescues the disrupted imprinting induced by somatic cell nuclear transfer in pigs.

Imprinting disorders induced by somatic cell nuclear transfer (SCNT) usually lead to the abnormalities of cloned animals and low cloning efficiency. Histone deacetylase inhibitors have been shown to improve gene expression, genomic methylation reprogramming and the development of cloned embryos, however, the imprinting statuses in these treated embryos and during their subsequent development remain poorly studied. In this study, we investigated the dynamics of H19/Igf2 methylation and transcription in porcine cloned embryos treated with trichostatin A (TSA), and examined H19/Igf2 imprinting patterns in cloned fetuses and piglets. Our results showed that compared with the maintenance of H19/Igf2 methylation in fertilized embryos, cloned embryos displayed aberrant H19/Igf2 methylation and lower H19/Igf2 transcripts. When TSA enhanced the development of cloned embryos, the disrupted H19/Igf2 imprinting was largely rescued in these treated embryos, more similar to those detected in fertilized counterparts. Further studies displayed that TSA effectively rescued the disrupted imprinting of H19/Igf2 in cloned fetuses and piglets, prevented the occurrence of cloned fetus and piglet abnormalities, and enhanced the full-term development of cloned embryos. In conclusion, our results demonstrated that aberrant imprinting induced by SCNT led to the abnormalities of cloned fetuses and piglets and low cloning efficiency, and TSA rescued the disrupted imprinting in cloned embryos, fetuses and piglets, and prevented the occurrence of cloned fetus and piglet abnormalities, thereby improving the development of cloned embryos. This study would have important implications in improving cloning efficiency and the health of cloned animals.