Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most infectious viral diseases of swine. Although Cluster of differentiation 163 (CD163) is identified as an essential receptor for mediating PRRS virus (PRRSV) infection, the important residues involved in infection on CD163 are still unclear. Therefore, it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs. In this study, we first generated immortalized porcine alveolar macrophage (IPAM) cell lines harboring 40-residues (residues 523–562, including R561 (arginine (R) at position 561)) deletion of CD163. PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection. We then generated cloned pigs carrying CD163- R561A (an arginine (R) to alanine (A) substitution at position 561 of CD163). PRRSV challenge experiments in porcine alveolar macrophages (PAMs) isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs. Through this study, we show that CD163 523–562 contains essential residues for mediating PRRSV infection, and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection. These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection. Furthermore, CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.  Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects.  However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research.  We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system.  After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus.  The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene.  Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting.  RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes.  Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P_24h=0.31, P_48h=0.96, P_72h=0.24, P_96h=0.17, and P_120h=0.38) on cell proliferation of PK15 cells.  These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment

With the rapid development of molecular biology and related disciplines, animal breeding has moved from conventional breeding to molecular breeding.  Marker-assisted selection and genomic selection have become mainstream practices in molecular breeding of livestock.  However, these techniques only use information from genomic variation but not multi-omics information,  thus do not fully explain the molecular basis of phenotypic variations in complex traits.  In addition, the accuracy of breeding value estimation based on these techniques is occasionally controversial in different populations or varieties.  Given the rapid development of high-throughput sequencing techniques and functional genome and dramatic reductions in the overall cost of sequencing, it is possible to clarify the interactions between genes and formation of phenotypes using massive sets of omic-level data from studies of the transcriptome, proteome, epigenome, and metabolome.  During livestock breeding, multi-omics information regarding breeding populations and individuals should be taken into account.  The interactive regulatory networks governing gene regulation and phenotype formation in diverse livestock population, varieties and species should be analyzed.  In addition, a multi-omics regulatory breeding model should be constructed.  Precision, population-personalized breeding is expected to become a crucial practice in future livestock breeding.  Precision breeding of individuals can be achieved by combining population genomic information at multi-omics levels together with genomic selection and genome editing techniques.  

    Porcine reproductive traits are characterized by low heritability, making improvement by traditional selective breeding rather difficult. Molecular breeding offers powerful approaches to overcome previous limitations and is expected to generate economic benefits via progress in pig breeding. Cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1) gene is a key enzyme of estradiol biosynthesis that plays an important role in the establishment of gestation and maintenance of pregnancy. In this study, the sequence and structure characteristics of the porcine CYP19A1 gene was analyzed and expression patterns of CYP19A1 in different tissues of adult female pigs were detected. Fourteen single-nucleotide polymorphisms (SNPs) in the exons and introns of porcine CYP19A1 were identified and genotyped using the Sequenom MassARRAY platform, after which the allele frequency of each SNP was analyzed. The association between CYP19A1 SNPs and litter size and piglet birth weight was assessed in a crossbred pig population (n=375). The expression pattern of CYP19A1 revealed that it was highly expressed in the ovary, spleen, and uterus and lowly expressed in the other tissues. Moreover, one SNP, rs341891833, was significantly associated with piglet birth weight during the multiparity period (P<0.01). We concluded that CYP19A1 could be used as a candidate molecular marker in breeding aimed at rapid improvement of the reproductive characteristics of pigs.  

Patatin-like phospholipase domain containing 5 (PNPLA5) is a neotype neutral lipase with dual activity of anabolism and catabolism in vitro and in vivo, which has a low mRNA expression level in humans and mice.  PNPLA5, which is localized to lipid droplets and required for efficient autophagy by optimal initiation, has been speculated to possess triglyceride hydrolase activity, and has been associated with low density lipoprotein cholesterol (LDL-C).  Above all, PNPLA5 is a relatively new gene, which is reported less about its biological function research, especially the function research in the rats is still blank.  In this study, we examined the spatiotemporal expression profile of PNPLA5 and found that it was expressed at low levels in most organs of Sprague Dawley (SD) rats, but was present at very high levels in the skin and testes.  To further determine the biological function of PNPLA5 in mammals, we generated PNPLA5-knockout SD rats using the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 system.  PNPLA5-null rats were viable, but showed a variety of phenotypic abnormalities, such as abnormal bleeding, and varied hematobiochemical parameters including increased serum total cholesterol (TC), triglycerides and high density lipoprotein cholesterol (HDL-C) level, and reduced LDL-C level, compared with wild-type control rats.  These data are consistent with an important role for PNPLA5 in lipid metabolism, providing a new target gene and animal model for treatment of cardiovascular diseases in the future.

Secreted frizzled-related protein 2 (SFRP2), a member of the SFRPs family, is associated with cell growth and differentiation in myogenesis. Our previous study suggested that SFRP2 was a potential target of microRNA (miRNA)-1/206, which was considered as myomiRs. To further explore the biological function and regulation mechanisms of the SFRP2 gene in porcine skeletal muscle development, we first analyzed the sequence structure of the porcine SFRP2 gene. Subsequently, we detected its tissue distribution in adult Tongcheng pigs (a Chinese indigenous breed) and investigated its dynamic expression in developmental skeletal muscle (13 prenatal and 7 postnatal time points) in Tongcheng pigs. An interaction analysis between SFRP2 and myomiRs was also performed. The results showed that the expression pattern of the SFRP2 varied greatly across diverse tissues. It exhibited abundant expression in prenatal skeletal muscle and peaked at 55 days post coitus (E55), and had a lower expression in postnatal skeletal muscle, indicating that the SFRP2 gene might affect porcine embryonic skeletal muscle development. Co-expression analysis revealed that the expression levels of SFRP2 correlated negatively with miRNA-1 (r=–0.570, P-value=0.009) and miRNA-206 (r=–0.546, P-value=0.013), but positively with SFRP1 (r=0.613, P-value=0.004). The bioinformatics analysis and dual luciferase assay verified that the SFRP2 was a putative target of miRNA-1/206 in pigs. Therefore, this study is helpful for understanding the biological function and molecular regulation of the SFRP2 gene during porcine skeletal muscle development.

Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig (WZSP) inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had been cultivated successfully, allogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients’ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4+ and CD8+ lymphocytes, and conducted hematoxylin-eosin (HE) and Masson’s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism (SNP) chips (60 000 SNPs). The result showed that there were no significant differences in graft skin, the plasma interleukin-2, 4, 10, CD4+ and CD8+, HE and Masson’s staining results between the allograft and autograft groups, and no immune rejection occurred on the allograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex (MHC) I and MHC II were homozygous which confirmed that immune antibody of the allograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the allograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successfully for the first time in the world. The test methods also provide a scientific basis for the identification of swine and mammal inbred lines.

MicroRNAs (miRNAs), evolutionarily conserved non-coding RNAs in length 21-24 bp, play a critical role in skeletal muscle development. In this study, to explore the function of mircoRNA-127 in porcine skeletal muscle development, eight tissue samples from adult pigs and longissimus muscle samples at 26 developmental stages were collected from Tongcheng and Landrace pigs. The spatial-temporal expression profiles of miRNA-127 were carried out using step-loop quantitative real-time PCR (stem- loop RT-PCR). To explore the molecular functions of miRNA-127, we predicted its target genes and performed functional annotation using bioinformatics methods. Results suggested that miRNA-127 was abundantly expressed in heart, ovary, uterus and spleen tissues and was weakly expressed in liver, lung, kidney and small intestine in both Tongcheng and Landrace pigs. And miRNA-127 showed significant expression differences in heart, ovary, spleen and uterus tissues between these two breeds. miRNA-127 basically kept at a relatively stable high level in middle and later embryonic stages and a low expression level in early embryonic stages and postnatal stages, but the expression levels of miRNA-127 were higher in Tongcheng pigs than in Landrace at most developmental stages. miRNA-127 potentially regulated 240 candidate genes. Results of Gene Ontology and KEGG pathway analysis indicated that these genes could be involved in many molecular functions and mechanisms, such as regulation of the force of heart contraction, regulation of transcription, regulation of T cell differentiation, MAPK signaling pathway and GnRH signaling pathway. Many significantly enriched GO terms and KEGG pathways were related to skeletal muscle development. This study will be helpful to understand the biological function for miRNA-127 and identify candidate gene associated with meat production traits in pigs.

Cellular retinoic acid-binding protein 1 (CRABP1) is a well-conserved member of cytosolic lipid-binding protein family. It is an important modulator of retinoic acid signaling. Long serial analysis of gene expression (LongSAGE) analysis suggested that CRABP1 gene was differentially expressed during prenatal skeletal muscle development in porcine. Here, we obtained the full-length coding region sequence and genomic sequence of the porcine CRABP1 gene and analyzed its genomic structures. Subsequently, we examined CRABP1 chromosome assignment using INRA-University of Minnesota 7 000 porcine radiation hybrid panel (IMpRH) and explored its tissue distribution in adult Tongcheng pigs and dynamical expression profiles in prenatal skeletal muscle (33, 65 and 90 days post coitus, dpc) from Landrace (lean-type) (described as L33, L65 and L90) and Tongcheng pigs (obese-type) (described as T33, T65 and T90). The CRABP1 gene was mapped to chromosome 7q11-q23 and closely linked to the microsatellite marker SWR1928. Quantitative real-time PCR showed that CRABP1 mRNA was highly expressed in lung and stomach, moderately expressed in placenta and uterus, and weakly expressed in other tissues. Moreover, CRABP1 gene was down-regulated during prenatal skeletal muscle development in both Landrace and Tongcheng pigs and it was expressed much higher in T33 than L33. Two single-nucleotide polymorphisms (SNPs) were detected by sequencing and mass spectrometry methods, allele frequency analysis indicated that g. 281 (G>A) and g. 2992 (G>A) were deviated from Hardy-Weinberg equilibrium in the Landrace and DLY (Duroc×(Landrace×Yorkshire)) pig breeds.

Type A spermatogonial stem cells are the only immortal diploid cells in the postnatal animal that undergo self-renewal through the lifetime of an animal and transmit genes to subsequent generations. In this paper, the generation and characterization of double-transgenic mice co-expressing the Escherichia coli appA gene and human MxA gene generated via the in vivo transfection of type A spermatogonial cells were reported for the first time. The dicistronic expression vector pcDNA-appA-MxA(AMP) and ExGen500 transfection reagent were injected into the testicular tissue of 7-d-old male ICR mice. The mice that underwent testismediated gene transfer were mated with wild-type female mice, and the integration and expression of the foreign genes in the offspring were evaluated. Transgenic mice that co-expressed appA and MxA showed a gene integration rate of 8.89% (16/180). The transgenic mice were environmentally friendly, as the amount of phosphorous remaining in the manure was reduced by as much as 11.1% by the appA gene (P<0.05); these animals also exhibited a strong anti-viral phenotype.

Beak trimming was a part of routine husbandry for poultry industry, which was the most efficient and economic way to reduce the incidence of cannibalism. However, the controversy about young chicken trimming still exits and it was drawing more concern recently along with the popular of animal behavior and welfare theory. To evaluate the influence of beak trimming on young chicken spleen development and find the key regulators of the spleen development, in this study, the electron microscope technique, flow cytometry and immunohistochemical techniques were used to investigate the effects of beak trimming stress on the spleen development. The result of electronic microscope showed that significant stress response could be found in chicken spleen cells after beak trimming. Cell volume became larger, chromatin of cell nuclear agglutinated conglobation, cytoplasm formed more vacuole, and mitochondria emerged vacuolization phenomenon in splenic lymphocyte after beak trimming. From the flow cytometry, the lymphocyte of spleen in the control group and experimental group were mainly in G1 phase, and the remaining small amount was in S and M phases. Of interest, the number of the lymphocyte which are in G1 phase within immune organ in the experimental group were more than that in the control group, but the number of cells in S and M phases were less than that in the control group. And beak trimming decreased the lymphocytes proliferation index of spleen. Inmmuohistochemical result showed that the beak trimming stress did not affect the expression sites of two apoptosis-related genes, Bcl-2 and Bax, in the spleen. But the expression level of Bcl-2 decreased while Bax increased in chicken immune organs after beak trimming. Furthermore, the beak trimming increased spleen cell apoptosis with a time-dependant style. Our findings may make a contribution to the active precaution of stress response.

Polyunsaturated fatty acids (PUFAs) are essential for normal growth in mammals, especially the ω-3 PUFAs, which play important roles in preventing several life-threatening diseases, such as coronary heart disease and diabetes. In this study, we aimed to investigate whether the sFat-1 gene from Caenorhabditis briggsae could be functionally expressed in transgenic pigs, and whether the transgenic could synthesize high quality ω-3 PUFAs endogenously. In this study, a gene construct consisting of CMV promoter and 1.9 kb cDNA of ω-3 fatty acid desaturase gene (sFat-1) from C. briggsae was injected into the male pronucleus of pig embryos by microinjection. The piglets were screened for the transgene by PCR, Southern blot and reverse transcription-PCR analysis. Pigs that give positive results were mated with wild-type pigs to produce the next generation and the transmission of transgene was examined by PCR analysis. Fatty acids compositions of various tissues in the transgenic pigs were then analyzed by gas chromatograph. A total of 878 embryos were transferred into 42 recipients, among which 29 successfully got pregnant and gave birth to a total of 162 piglets, and 8 of them were identified to be transgenic. Fatty acid compositions in the transgenic pigs were altered, and the levels of ω-6: ω-3 ratios were decreased from 14.53 in the control to 2.62 in Fat-1 transgenic pigs. A number of primary sFat-1-transgenic pigs were bred in this study, which lays the foundation for cultivation of new varieties of transgenic pigs.