猪繁殖与呼吸综合征（Porcine reproductive and respiratory syndrome, PRRS）是猪传染性最强和致死性最高的病毒性疾病之一。尽管分化抗原簇163 (Cluster of differentiation 163, CD163) 蛋白被确定为介导PRRS病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）感染的必需受体，但CD163参与感染的重要氨基酸残基仍不清楚。鉴定这些关键残基对于研究PRRSV的感染机制和制备抗PRRSV的基因编辑猪具有重要意义。对CD163结构的分析表明，CD163 SRCR5结构域内的配体结合口袋(ligand-binding pocket, LBP)(位于CD163的第487位到499位氨基酸)和loop 5–6（位于CD163的第544位到570位氨基酸）可能参与了PRRSV的感染。CD163 LBP位点特异性编辑猪可以完全抵抗PRRSV感染，但loop 5-6的破坏或缺失是否能抑制PRRSV的感染尚未报道。R561（第561位的精氨酸（R））位于loop 5-6内，R561A突变的PK-15细胞可以显著增强对PRRSV的抗感染能力，但定点编辑CD163 R561的猪肺泡巨噬细胞（porcine alveolar macrophages, PAMs）（PRRSV感染的靶细胞）或CD163 R561编辑猪能否抑制PRRSV的感染仍不清楚。这项研究中，我们首先利用CRISPR/Cas9介导的片段删除技术制备了CD163缺失40个氨基酸残基的永生化猪肺泡巨噬细胞（immortalized porcine alveolar macrophage, IPAM）细胞系，这40个残基位于CD163的第523位到562位氨基酸，这其中包含了R561和部分loop 5–6结构，该细胞系被命名为IPAM-CD163^△523-562。病毒感染实验表明，IPAM-CD163^△523-562可以完全抵抗 PRRSV的感染。同时，我们利用CRISPR/Cas9介导同源重组技术制备了携带CD163-R561A（CD163的561位精氨酸（R）被替换为丙氨酸（A））的基因编辑克隆猪，并分离得到原代CD163-R561A PAMs。PRRSV攻毒实验结果表明，与野生型的CD163-R561 PAMs相比，CD163-R561A PAMs 对PRRSV的易感性显著降低。以上研究结果表明，CD163的第523位到562位氨基酸中含有介导PRRSV感染的必需氨基酸残基，CD163 R561参与了PRRSV的感染过程，但它不是感染所必需的。这些位点可以作为了解PRRSV感染机制的新靶点，CD163-R561A猪也可作为培育抗PRRSV猪群体的育种材料。

外源基因在转基因动物和细胞中稳定、高效的表达，对于基因功能的研究和生物反应器的建立至关重要。动物基因组中的友好基因座能使外源基因高效稳定的表达且无副作用，但目前猪基因组中可供外源基因安全、高效定点整合的基因座相对较少，限制了多转基因猪的研究和发展。本研究提出了一种将猪I型胶原α1链（collagen type I alpha 1 chain，COL1A1）基因座作为友好基因座表达外源基因的新策略。利用CRISPOR软件设计了一对靶向COL1A1基因终止密码子的sgRNA，并连接到CRISPR/Cas9表达载体pX330中；同时合成了一个不含启动子、左右同源臂各长为900 bp的2A-GFP供体载体，然后共转染猪肾上皮细胞（porcine kidney epithelial，PK15)，胎儿成纤维细胞（porcine embryonic fibroblast，PEF）以及回肠上皮细胞（porcine intestinal epithelial，IPI-2I）3种细胞。电转染48 h后观察3种细胞的荧光情况，72 h后通过流式细胞术分选绿色荧光蛋白阳性的细胞，并通过荧光定量、蛋白质印记、转录组测序和CCK8实验在不同水平上评估COL1A1基因作为猪基因组外源基因定点整合位点的安全性。结果表明，共转染sgRNA和供体载体之后，可以分别在猪3种不同细胞中观察到绿色荧光，说明同源重组介导的定点整合系统可以在猪基因组中很好的发挥作用。qPCR结果显示，GFP敲入对3种细胞COL1A1基因mRNA的表达并无显著影响（PK15、PEF和IPI-2I细胞中的P值分别为0.29、0.66和0.20）。同样，蛋白质印记结果显示，GFP阳性细胞与野生型细胞COL1A1蛋白的表达并无显著差异（PK15、PEF和IPI-2I细胞中的P值分别为0.64、0.48和0.80)。转录组测序结果显示，GFP阳性PEF细胞与野生型PEF细胞的转录组显著正相关（P<2.2e-16），表明GFP敲入没有改变内源性基因的整体表达。CCK8实验表明，GFP敲入对PK15细胞增殖并无显著影响（24 h，48 h，72 h，96 h，120 h的P值分别为0.31，0.96，0.24，0.17和0.38）。上述结果表明，COL1A1基因可作为猪基因组中外源基因定点整合的友好基因座，可广泛应用于家畜育种和生物医学模型的建立。本研究首次鉴定出了COL1A1基因可以作为猪基因组中的友好基因座。

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.