[Progress on abnormal development of cloned pigs generated by somatic cell transfer nuclear].

Cloning, also known as somatic cell nuclear transfer (SCNT), is an asexual reproduction technique that reprograms differentiated cells to the totipotent state, and generates offspring with a genotype identical to the donor cells. Pig cloning technique holds great promise for propagating excellent breeding boars, generating genetically modified pigs, protecting rare and endangered pigs and studying the mechanisms of somatic cell nucleus reprogramming. However, cloned pigs suffer from various developmental defects, including low birth rate, low birth weight, and high stillbirth occurrence, neonatal mortality and congenital malformations, which severely hamper their applications. Errors in epigenetic reprogramming of donor nucleus are considered as the main causes of low cloning efficiency and abnormal embryonic development in cloned embryos and animals. However, most studies to correct the errors in epigenetic reprogramming of cloned pig embryos have not substantially improved the birth and survival rates of cloned pigs. In this review, we summarize the abnormal phenotypes, causes of abnormal development of cloned pigs and effective methods for improving pig cloning efficiency, thereby providing a reference for the future research to improve the development and survival rates of cloned pig embryos and cloned pigs.

Transcriptome heterogeneity of porcine ear fibroblast and its potential influence on embryo development in nuclear transplantation.

There is heterogeneity among donor cells of the same source. Many studies have shown that donor cell affects the efficiency of somatic cell nuclear transfer (SCNT). However, the potential influence of donor cell heterogeneity on the efficiency of nuclear transplantation were rarely analyzed at the single-cell level. In this study, single-cell transcriptome sequencing was performed on 52 porcine ear fibroblasts randomly selected from the same source to compare their gene expression patterns. The results showed that 48 cells had similar gene expression patterns, whereas 4 cells (D11_1, D12_1, DW61_2, DW99_2) had significantly different gene expression patterns from those of other cells. There were no two cells with identical gene expression patterns. The gene expression patterns of D11_1, D12_1, DW61_2 and DW99_2 were analyzed, using the 48 cells with similar gene expression patterns as controls. Firstly, we used the R language statistics to select the differentially expressed genes in the 4 single cells, and identified the top 50 most significant differentially expressed genes. Then GO enrichment analysis and KEGG pathway analysis were performed on the differentially expressed genes. Enrichment analysis revealed that the main molecular functions of the differentially expressed genes included energy metabolism, protein metabolism and cell response to stimulation. The main pathways from KEGG enrichment were related to cell cycle, cell metabolism, and DNA replication. Finally, based on the above results and in consideration with the SCNT research progress, we discussed the potential effects of differential gene expression patterns of the 4 single cells on the embryonic development efficiency of nuclear transplantation. This study revealed transcriptional heterogeneity of porcine ear tissue fibroblasts and provided an effective method to analyze elite donor cells, thereby providing new ideas on improving the cloning efficiency of SCNT.

[Recent developments in enhancing the efficiency of CRISPR/Cas9- mediated knock-in in animals].

Gene-editing technology can artificially modify genetic material of targeted loci by precise insertion, deletion, or replacement in the genomic DNA. In recent years, with the developments of zinc-finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) technologies, such precise modifications of the animal genomes have become possible. Although gene-editing tools, such as CRISPR/Cas9, can efficiently generate double-strand breaks (DSBs) in mammalian cells, the homology-directed repair (HDR) mediated knock-in (KI) efficiency is extremely low. In this review, we briefly describe the current development of gene-editing tools and summarize the recent strategies to enhance the CRISPR/Cas9- mediated KI efficiency, which will provide a reference for the generation of human disease models, research on gene therapy and livestock genetic improvement.

[Advances in assay for transposase-accessible chromatin with high-throughput sequencing].

Assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) was developed in 2013. It has the advantages of more convenient operation and higher efficiency for DNA recovery than DNase I hypersensitive site sequencing (DNase-seq) and micrococcal nuclease sequencing (MNase-seq). ATAC-seq currently is the most popular technique of genome-wide mapping for chromatin accessibility. It provides information on binding regions of transcription factors and nucleosome localization on the chromatin. Thus, ATAC-seq is of great significance for studying the epigenetics and molecular mechanisms in chromatin structure. In this review, we compare the advantages and disadvantages of multiple techniques for profiling chromatin accessibility, and summarize the principles, main process, development and applications of ATAC-seq. We hope this review will provide a reference for study of genome-wide mapping for chromatin accessibility, identification of cis-regulatory elements, and dissection of the epigenetic and genetic regulatory networks using the ATAC-seq technology in eukaryotes.

[Advances in epigenetic reprogramming of somatic cells nuclear transfer in mammals].

Somatic cell nuclear transfer (SCNT) is the only reproductive engineering technique that can confer genomic totipotency on somatic cell. SCNT is of great significance for animal germplasm conservation, animal husbandry development, and biomedical research. Although many research advances have been made in this technology, the developmental rate of SCNT mammalian embryos is very low, which seriously limits the application of SCNT in animal husbandry and biomedicine. The primary reason for the low efficiency of cloned embryos is somatic cell reprogramming errors or incomplete reprogramming. These errors or incompleteness present as the abnormal expression of imprinted gene Xist, abnormal DNA methylation, and abnormal histone modification. In this review, we summarize the main factors that influence the low development efficiency of mammalian cloned embryos to provide theoretical reference for the research and practice of improving somatic cell cloning efficiency.

[Heterogenous expression and enzymatic property analysis of microbial-derived pectinases in pig PK15 cells].

As one of plant cell wall components, pectin is the main anti-nutritional factor in livestock and poultry feeds and has an adverse effect on utilization efficiency of feed energy and nitrogen. Pectinases, which are widely found in microorganisms such as bacteria, yeast and filamentous fungi in nature,can improve feed efficiency by relieving the anti-nutritional effect of pectin through promoting the hydrolysis reaction of feed pectin. To explore the feasibility of expressing microbial-derived pectinase genes in pig cells, we introduced microbial-derived pectinase genes pg5a, pgI, pga3A, and pgaA into porcine PK 15 cells by lipofection for heterogenous expression. Enzymatic activities of the pectinases encoded by these genes were analyzed using the 3,5 dinitrosalicylic acid (DNS) method. Results showed that all four pectinase genes were able to be transcribed into mRNAs in porcine PK 15 cells, but only pg5a and pgI were adapted to the porcine cell expression system. Among them, the maximum activity of pectinase PG5A was 0.95 U/mL, the optimum pH was pH 4.0, and the enzymatic activity was maintained above 46% in the range of pH 4.6 to 6.0. Pectinase PGI obtained the highest enzymatic activity at pH 5.0, which was 0.30 U/mL, and maintained more than 35% of the activity in the range of pH 4.0 to 6.0. The results of digestive protease tolerance test showed that PG5A and PGI were highly resistant to pepsin and trypsin. After treatment with 1 mg/mL pig pepsin for two hours, the residual enzymatic activities of PG5A and PGI were 76% and 71%, respectively. And after two hours treatment with 1 mg/mL of pig trypsin, the remaining enzymatic activities of PG5A and PGI were 44% and 93%, respectively. In summary, pectinase PG5A and PGI can be effectively expressed in pig cells, and have strong tolerance to pig intestinal pH environment and digestive proteases. Therefore, both pg5a and pgI can be used as candidate genes for production of transgenic pigs.

[Histone H3K27me3 in the regulation of skeletal muscle development].

Histone methylation is a modification which occurs in the N-terminal peptide chains of the histone nucleosome. The 4th, 9th, 27th, 36th and 79th lysines in N-terminal peptide chain of histone H3 are hot spots for this modification, including mono-, di-, and tri-methylation. H3K27me3 is the tri-methylation modification on histone H3 lysine 27, which mainly functions as a transcriptional repressor regulating skeletal muscle development. Studies have shown that H3K27me3 can finely regulate skeletal muscle proliferation, including the level and duration of skeletal muscle development by specifically binding to myogenic regulatory factors (e.g., MyoD, MyoG, etc.), cell cycling regulators, and epigenetic regulators including lncRNA and miRNA. In this review, we introduce the types and mechanisms of histone methylation and de-methylation of H3K27. We also summarize how H3K27me3 functions in the proliferation and differentiation of skeletal muscle cell. This review will contribute to the comprehension of the function of H3K27me3 in regulating skeletal muscle development and provide reference for further improving our understanding of mammalian muscle.

[MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts].

There are two major pathways, homology-directed repair (HDR) and nonhomologous end joining (NHEJ), involved in double-strand break (DSB) repair. Single-stranded oligodeoxyribonucleotide (ssODN)-mediated homologous recombination repair is commonly used for animal site-directed genome editing, with great scientific and practical value. To improve ssODN-mediated HDR efficiency in the pig genome, we investigated the effect and molecular mechanism of mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor PD0325901 on the HDR efficiency in porcine fetal fibroblasts (PFFs). The results showed that PD0325901 obviously increased the percentage of G2 and S phase cell populations and reduced the cell population ratio in the G1 phase of PFFs, and promoted the expression of HDR repair factor. At the optimal concentration of 250 nmol/L, PD0325901 increased the repair efficiency of ssODN-mediated GFP reporter vector by 58.8% and the directed editing efficiency of PFF DMD and ROSA26 locus by 48.16% and 17.64%, respectively. The results show that MEK inhibitor PD0325901 significantly promotes the efficiency of ssODN-mediated homologous-directed repair in the porcine genome, thus offering a new idea to generate genetically modified pigs more effectively.

ß-Glucanase specific expression in the intestine of transgenic pigs.

Producing heterologous enzymes in the animal digestive tract to improve feed utilization rate is a new research strategy by transgenic technology. In this study, transgenic pigs specifically expressing ß-glucanase gene in the intestine were successfully produced by somatic cell nuclear transfer technology in order to improve digestibility of dietary ß-glucan and absorption of nutrients. The ß-glucanase activity in the intestinal juice of 4 transgenic pigs was found to be 8.59 ± 2.49 U/mL. The feeding trial results showed that the crude protein digestion of 4 transgenic pigs was significantly increased compared with that of the non-transgenic pigs. In order to investigate the inheritance of the transgene, 7 G1 transgenic pigs were successfully obtained. The ß-glucanase activity in the intestinal juice of 7 G1 transgenic pigs was found to be 2.35 ± 0.72 U/mL. The feeding trial results showed the crude protein digestion and crude fat digestion were significantly higher in 7 G1 transgenic pigs than in non-transgenic pigs. Taken together, our study demonstrated that the foreign ß-glucanase expressing in the intestine of the transgenic pigs could reduce the anti-nutritional effect of ß-glucans in feed. In addition, ß-glucanase gene could be inherited to the offsprings and maintain its physiological function. It is a promising approach to improve feed utilization by producing transgenic animals.

[Effects of RNA interference on the porcine NHEJ pathway repair factors on HR efficiency].

Non-homologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells. It inhibits the efficiency of homologous recombination (HR) by competing for DSB targets. To improve the efficiency of HR in porcine fetal fibroblasts (PFFs), several RNA interference (RNAi) systems were designed to knockdown NHEJ key molecules, such as polynucleotide kinase/phosphatase (PNKP), DNA ligase IV (LIG4) and NHEJ1. The results show that siRNA significantly knocked down LIG4, PNKP and NHEJ1 expression. Suppression of PNKP dramatically increased the efficiency of single-strand annealing (SSA), double-strand DNA (dsDNA) and single-strand DNA (ssODN) mediated homology-directed repair (HDR) by 55.7%, 37.4% and 73.1% after transfected with the SSA-GFP reporter, HDR-GFP system or ssODN-GFP system, respectively; whereas knockdown of LIG4 and NHEJ1 repair factors significantly increased dsDNA or ssODN-mediated HDR efficiency by 37.5% and 76.9%, respectively.

[Progress and application of genome-edited pigs in biomedical research].

Genome editing technologies (GETs) can precisely alter the genomic sequences and modify the genetic information at the target site of an organism. Since the beginning of the 21st century, the GETs, including zinc finger nucleases (ZFN), transcription-activating-like receptor factor (TALEN), and clustered regularly interspaced short palindromic repeats/Cas endonucleases (CRISPR/Cas), have been successively developed. The GETs can easily engineer the targeted genomic site of animals to exhibit a desired phenotype(s), thereby providing valuable tools in biomedical research. The pigs are closely related to human, in terms of similarities in physiological properties and pathogenic characters. Thus, pigs have been used as important animal models in studies of human disease, xenotransplantation, and humanized organs regeneration. In this review, we summarize the development of the three GETs, research progress of genome-edited pigs as disease models and organ donors for xenotransplantation, and the prospects of their applications in future biomedical research.

Application of genomic selection in farm animal breeding.

Genomic selection (GS) has become a widely accepted method in animal breeding to genetically improve economic traits. With the declining costs of high-density SNP chips and next-generation sequencing, GS has been applied in dairy cattle, swine, poultry and other animals and gained varying degrees of success. Currently, major challenges in GS studies include further reducing the cost of genome-wide SNP genotyping and improving the predictive accuracy of genomic estimated breeding value (GEBV). In this review, we summarize various methods for genome-wide SNP genotyping and GEBV prediction, and give a brief introduction of GS in livestock and poultry breeding. This review will provide a reference for further implementation of GS in farm animal breeding.

Effects of parameters, plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators.

To obtain an ideal transfection efficiency of porcine fetal fibroblasts, fluorescence activated cell sorting (FACS) was used to optimize parameters for transfection of porcine fetal fibroblasts (PFFs) with ECM? 830, NEPA 21 and Nucleofector? 2b in different conditions such as electroporation parameters, plasmid dosages and topological structures. The results show that the optimum poring pulse parameter of NEPA 21 is voltage 200 V, continuous 3 ms, interval 50 ms, 3 times, voltage attenuation range of 10%; and the transfection efficiency of Nucleofector? 2b is highest under U-023 program. Under the optimum conditions, FACS analysis demonstrates that Nucleofector? 2b and ECM? 830 have the highest transfection efficiency when transfecting 10 µg supercoiled plasmids into PFFs, and 8 µg for NEPA 21. Supercoiled plasmids show higher transfection efficiencies than linearized plasmids. Moreover, Nucleofector? 2b has the highest transfection efficiency among the three electroporation instruments. This study paves the way to generate transgenic or gene editing pigs with high efficiency.

Advances in site-specific integration of transgene in animal genome.

The traditional transgenic technologies, such as embryo microinjection, transposon-mediated integration, or lentiviral transfection, usually result in random insertions of the foreign DNA into the host genome, which could have various disadvantages in the establishment of transgenic animals. Therefore, a strategy for site-specific integration of a transgene is needed to generate genetically modified animals with accurate and identical genotypes. However, the efficiency for site-specific integration of transgene is very low, which is mainly caused by two issues. The first one is the low efficiency of inducing double-strand break (DSB) at the target site of host genome in the initial process. The second one is the low efficiency of homologous recombination repair (HDR) between the target site and the donor plasmid carrying homologous arm and foreign genes. HDR is the most common mechanism for site-specific integration of a transgene. DSBs can stimulate DNA repair mainly by two competitive mechanisms, HDR and nonhomologous end joining (NHEJ). Hence, activation of HDR or inhibition of NHEJ can promote the HDR in the integration processes, thereby optimizing a specific targeting of the transgene. In this review, we summarize the recent advances in strategies for improving the site-specific integration of foreign transgene in transgenic technologies.

Improvement of anti-nutritional effect resulting from ß-glucanase specific expression in the parotid gland of transgenic pigs.

ß-Glucan is the predominant anti-nutritional factors in monogastric animal feed. Although ß-glucanase supplementation in diet can help to eliminate the adverse effects, enzyme stability is substantially modified during the feed manufacturing process. To determine whether the expression of endogenous ß-glucanase gene (GLU) in vivo can improve digestibility of dietary ß-glucan and absorption of nutrients, we successfully produced transgenic pigs via nuclear transfer which express the GLU from Paenibacillus polymyxa CP7 in the parotid gland. In three live transgenic founders, ß-glucanase activities in the saliva were 3.2, 0.07 and 0.03 U/mL, respectively, and interestingly the enzyme activities increased in the pigs from 178 days old to 789 days old. From the feed the amount of gross energy, crude protein and crude fat absorbed by the transgenic pigs was significantly higher than the non-transgenic pigs. Meanwhile the moisture content of the feces was significantly reduced in transgenic pigs compared with the non-transgenic pigs. Furthermore, in all positive G1 pigs, ß-glucanase activity was detectable and the highest enzyme activity reached 3.5 U/mL in saliva. Also, crude protein digestion was significantly higher in G1 transgenic pigs than in control pigs. Taken together, our data showed that the transgenic ß-glucanase exerted its biological catalytic function in vivo in the saliva, and the improved performance of the transgenic pigs could be accurately passed on to the offspring, indicating a promising alternative approach to improving nutrient availability was established to improve utilization of livestock feed through transgenic animals.

Establishment of porcine Xist knockout model using CRISPR/Cas9 system.

Somatic cell nuclear transfer technique has great applications in livestock breeding, production of genetically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this technique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that it may directly or indirectly affects cloning efficiency. In this study, multiple sgRNAs were designed based on the CRISPR/Cas system, and two sites (Target 3 and Target 4) whose mutation efficiency were 1% and 3% at the cellular level were selected. We successfully knocked out Xist with 100% efficiency by microinjecting sgRNAs for Target 3 and Target 4 in embryo. Finally, 6 cloning piglets were born including two Xist-fully-knockout piglets. The follow-up studies on increasing cloning efficiency can be carried out based on the Xist-knockout model.

[Placental developmental defects in cloned mammalian animals].

The cloning technique, also called somatic cell nuclear transfer (SCNT), has been successfully established and gradually applied to various mammalian species. However, the developmental rate of SCNT mammalian embryos is very low, usually at 1% to 5%, which limits the application of SCNT. Placental developmental defects are considered as the main cause of SCNT embryo development inhibition. Almost all of SCNT-derived mammalian placentas exhibit various abnormalities, such as placental hyperplasia, vascular defects and umbilical cord malformation. Mechanistically, these abnormalities result from failure of establishment of correct epigenetic modification in the trophectoderm genome, which leads to erroneous expression of important genes for placenta development-related, particularly imprinted genes. Consequently, aberrant imprinted gene expression gives rise to placental morphologic abnormalities and functional defects, therefore decreases developmental competence of cloned embryos. Currently, although numerous methods that can improve the developmental ability of SCNT-derived embryos have been reported, most of them are unable to substantially enhance the success rate of SCNT due to failure to eliminate the placental development defects. In this review, we summarize placental abnormalities and imprinted gene expression in mammalian cloning, and propose directions for the future research aiming to improve the cloning efficiency.

[Genetic diversity based on swine leukocyte antigen complex mi-crosatellites(SLA-MS) in five pig populations].

The genetic diversity of swine leukocyte antigen complex (SLA) was studied among Guangdong local pigs, Huanan wild boars (S.s. chirodontus) and introduced pigs, which aimed at providing a theoretical foundation for further pig anti-disease resistance breeding. Pietrain pigs, Duroc pigs, Large black-white pigs, Lantang pigs, and Huanan wild boars were genotyped by employing 18 microsatellites in swine leukocyte antigen complex (SLA-MS). The result showed that the average diversity in SLA II was higher (He=0.628, PIC=0.581) than that in SLA I (He=0.530, PIC=0.474) and in SLA III (He=0.526, PIC=0.458). The molecular diversity indices (MDI) of Huanan wild boars was the highest(0.716), followed by Lantang pigs (0.614), Large black-white pigs (0.559), Pietrain pigs (0.550) and Duroc pigs (0.507). As a whole, the genetic diversity of Huanan wild boars was the highest over Guangdong native pigs and introduced pigs. Large black-white pigs and Duroc pigs had ever happened a severe bottleneck by comparison with the Garza-Williamson index (GWI) in Huanan wild boar. From the genetic distance, one clade was that Lantang pigs were first clustered with Huanan wild boar, and then grouped together with Large black-white pigs; another clade was that Pietrain pigs were independently clustered with Duroc pigs in the NJ tree. The results would establish the foundation for pig conservation of germplasm resource, disease resistance breeding, and multiplicative strains.

Improving phytase enzyme activity in a recombinant phyA mutant phytase from Aspergillus niger N25 by error-prone PCR.

The mutant acid phytase (phyA ( m )) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NP(ep)-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyA ( ep ) gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower k (m) value than that of PP-NP(m)-8 (P < 0.05). In addition, the overall catalytic efficiency (k (cat)/k (m)) of the mutant one was 84% higher (P < 0.05) than that of PP-NP(m)-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.

Molecular characterization of caveolin-1 in pigs infected with Haemophilus parasuis.

Caveolin-1 (Cav1) plays a critical role in the invasion of pathogenic microbes into host cells, yet little is known about porcine Cav1. In this study, we provide the molecular characterization of Cav1 in pigs following stimulation with LPS/polyinosinic-polycytidylic acid as well as during infection with Haemophilus parasuis. The porcine Cav1 gene is 35 kb long and is located at SSC18q21; two isoforms (Cav1-α and Cav1-ß) are produced by alternative splicing. Three point mutations were identified in the coding region of the gene, two of which were significantly associated with nine immunological parameters in Landrace pigs, including the Ab response against porcine reproductive and respiratory syndrome virus and lymphocyte counts. Promoter analysis indicated that NF-κB activates both Cav1 transcripts, but the forkhead gene family specifically regulates Cav1-ß in the pig. Porcine Cav1 is expressed ubiquitously, with Cav1-α more abundantly expressed than Cav1-ß in all tissues investigated. Basal expression levels of Cav1 in PBMCs are relatively similar across different pig breeds. LPS and polyinosinic-polycytidylic acid markedly induced the expression of Cav1 in porcine kidney-15 cells in vitro, likely through NF-κB activation. Pigs infected with H. parasuis exhibited decreased expression of Cav1, particularly in seriously impaired organs such as the brain. This study provides new evidence that supports the use of Cav1 as a potential diagnostic and genetic marker for disease resistance in animal breeding. In addition, our results suggest that Cav1 may be implicated in the pathogenesis of Glasser's disease, which is caused by H. parasuis.