[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.

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.

[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.

[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.

[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.

[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.

[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).

[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.

β-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 β-glucanase gene in the intestine were successfully produced by somatic cell nuclear transfer technology in order to improve digestibility of dietary β-glucan and absorption of nutrients. The β-glucanase activity in the intestinal juice of 4 transgenic pigs was found to be 8.

[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.

[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.

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).

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.

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.

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.

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

β-Glucan is the predominant anti-nutritional factors in monogastric animal feed. Although β-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 β-glucanase gene (GLU) in vivo can improve digestibility of dietary β-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.

[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.

Characterization of novel EGs reconstructed from Bacillus subtilis endoglucanase.

Bacterial cellulases have taken on satisfactory application performance and economic value in detergent industry. Neutral endoglucanase (EG1) gene was cloned from Bacillus subtilis and expressed Pichia pastoris in our previous study. Redesigned endoglucanases enhanced cellulase domain, added and deleted carbohydrate-binding module (CBM), named EG2, EG3, and EG4, respectively, were constructed in this study.

[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.

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.

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-β) are produced by alternative splicing.

Genomic organization and polymorphisms detected by denaturing high-performance liquid chromatography of porcine SLC11A1 gene.

SLC11A1 (also known as Natural Resistance Associated Macrophage Protein1, NRAMP1) plays a crucial role in resistance of inbred mice to infection with several intracellular pathogens such as Mycobacterium, Leishmania and Salmonella. In this study, PCR amplification and sequencing were performed to obtain the genomic organization and sequence of porcine SLC11A1 gene by comparative genomic analysis. Results showed that porcine SLC11A1 gene consists of 15 exons and 14 introns, which is consistent with that of mice and human.

Study on the association between estrogen receptor gene (ESR) and reproduction traits in Landrace pigs.

To study the potential use of estrogen receptor gene (ESR) as a genetic marker to improve the reproductive traits of pigs, the genotypes of the ESR PCR product digested by Pvu II were determined in 2,239 litters from 612 Landrace sows. The data of the first, second, and later parities were separately evaluated. Although the frequency of the B allele was much lower than that of the A allele, likelihood ratio test showed that the gene frequencies were in Hardy-Weinberg equilibrium.

[Sequence and polymorphism analysis of porcine hormone-sensitive lipase gene 5'-UTR and exon I].

Hormone-sensitive lipase (HSL) is the key enzyme responsible for the mobilization of free acids from adipose tissue, and it is also the most important enzyme that affect fat deposition. In this paper, the porcine hormone-sensitive lipase gene 5'-UTR and exon I were sequenced. The sequence number in GenBank are AY332499, AY332497, AY332504, AY332505.

[Using VCE4.0 package to estimate genetic parameters on growth traits in Landrace].

It is an accurate and quick methods to apply VCE4.0 to estimating genetic parameter. It makes full use of all information, analyzing selection and culling effects.