As a new technology to improve agricultural technology and to raise agricultural production efficiency, GM technology from livestock production chain is an inevitable technosphere and it should be actively responded. In view of the coexist of safety and risk of GM food, animal feed and animal products, this paper expounded the concepts of GM organisms and GM animal, and summarized the attitudes of countries around the world and its consumers to GM technology together with its products. Among of this, the European Union(EU) shows prudent attitude to the development of GM food, animal feed and GM animal. EU startes early in terms of relevant identification and legislation and constantly improves the legislation following the progress of relevant technologies. EU claims the GM modified threshold of food and feed is 0.9%, while Australia and New Zealand are 1%. However, Japan and Korea show no request. The United States, Canada and Argentina adapt voluntarily to the identification of GM products. The current legislation of China focuses on the safety assessment of GM products, but they do not define the management and identification to the final products. Otherwise, this paper summarized all the work carried out in transgenic animal traceability in China as following: The first, transgenic animal database based on genetic modification and traceability network platform(www.gmanimal.com.cn) have been constructed basically. Secondly, the whole technical route of identification of transgenic animal product traceability is proposed. Thirdly, the model system of transgenic animals (pig) and their product traceability is developed by using net and network database technologies, and can realize whether genetically modified feeds and (or) genetically modified animals and their products tracking from origin to table and tracing against the above direction, and it is got ready to fulfill government's regulation and consumer's right to know and to choice what they want from technologies

The production and quality of pork is closely related to muscle development. Muscle development needs to undergo several steps: The formation of prenatal muscle fiber, the differentiation and enlongation of postnatal muscle fiber and the renewal of adult muscle. The myogenesis process is controled by a complicated network, including tanscriptional, post-transcriptional regulation and several pathways. Starting from the process of porcine myogenesis, we will mainly discuss the molecular mechanism of myogenesis regulation and the transgenetic pigs for pork improvement in this review, which will provide the basis for cultivating pigs with high lean meat percentage and obtianing pork of high quality

Polyamines are ubiquitous organic cations of low molecular weight. The role of polyamines in the animal digestion, nutrient metabolism and normal growth is considered. We cloned polyamine metabolism-related genes polyamine-modulated factor 1(PMF1), arginine decarboxylase(ADC), ornithine decarboxylase(ODC), ornithine aminotransferase(OAT), ornithine decarboxylase antizyme 1~3(OAZ1, OAZ2 and OAZ3), their fragment length were 1 602(GenBank No. EU545649), 1 422(GenBank No. EU534186), 1 465(GenBank No. EU404089), 874(GenBank No. EU545195), 678(GenBank No. EU545196), 667(GenBank No. EU545197) and 703 bp(GenBank No. EU534185), respectively, with the open reading frame(ORF) of 1 380, 1 383, 1 313, 681, 568, 559 and 615 bp and, coding amino acids of 460, 461, 439, 227, 189, 186 and 205, respectively. The results of comparative analysis illustrated that the porcine ADC encoding of amino acid sequence shared 89.7% and 87.4% similar to that of human (Homo sapiens) and mouse(Mus musculus) respectively; the porcine ODC one shared 93.1% , 89.8% and 90.5% similar to that of human, mouse and rat respectively; the porcine OAT one shared 91.8%, 90.5% and 89.8% similar to that of human, mouse and rat (Rattus norvegicus) respectively; the porcine OAZ1 one shared 92.5%, 85.1% and 84.3% similar to that of human, mouse and rat respectively; the porcine OAZ2 one shared 98.1%, 97.9% and 97.2% similar to that of human, mouse and rat respectively; the porcine OAZ3 one shared 90.4%, 87.5% and 84.2% similar to that of human, mouse and rat respectively; the porcine PMF1 one shared 84% and 70.9% similar to that of human and mouse respectively. The analysis of the semi-quantitative RT-PCR showed that, after weaning, ADC mRNA increased significantly in appendix and rectal, while decreased slightly in other tissues; ODC mRNA increased significantly in appendix and rectal, while decreased slightly in jejunum, ileum and colon; OAT mRNA increased significantly in renal, while decreased slightly in duodenum, jejunum and ileum; OAZ1 mRNA increased in different magnitude, except in stomach; OAZ2 mRNA increased in different magnitude, especially in the appendix; OAZ3 mRNA increased in different magnitude in the intestinal tissues; PMF1 mRNA was similar to the (AZ) family in jejunum, ileum and colonic, but decreased slightly. These results suggest that polyamine are involved in the effect of the growth, development, mature, adaptability and repair of injured in piglet

Thyroid hormone responsive SPOT14(THRSP), which is a nuclear gene induced by thyroid hormone, expressed predominately in adipose tissue and liver. It is involved in transcriptional regulation of rate-limiting enzymes in lipogenic pathway. It plays an important role on the regulation of animal lipogenesis. In order to further study the function of SPOT14 gene(GenBank accession No. JF951726)， the full cDNA was inserted into the expression vector pET28α(+), and the recombinant expression plasmid pET-28α(+)-S14 was transformed into Escherichia coli BL21(DE3). SDS-PAGE analysis of the IPTG induced expression of the pET-28α(+)-S14 showed that the weight of the target protein was about 20.91 kD, the fusion protein was mainly expressed in the inclusion body, and the best induction condition was 1 mmol/L IPTG for 5 h on 37℃. The fused protein with His-tag was purified by 6xHis Ni-NTA affinity chromatography, and a 21 kD protein was detected by Western blot. This study showed that the recombinant vector pET-28α(+)-S14 can express fused protein in BL21, which paves a way for further study on the gene function and protein function of SPOT14 and the regulation of lipometabolism mechanism

The efficiency of somatic cell nuclear transfer (SCNT) is low, which may be related to the poor quality of the oocyte at the beginning of maturation. Porcine oocytes are commonly recovered from ovaries of slaughtered animals, which indicates that oocytes may be at various stages of the estrous cycle. Therefore, selection of high quality oocytes is necessarry for SCNT. This study was conducted to examine the effect of brilliant cresyl blue(BCB) staining and trichostain A(TSA) on the development of SCNT embryos in Sus scrofa ussuricus. Cumulus-oocyte complex(COCs) were stained with BCB, then divided into three groups: Control(without BCB staining), BCB+ group(coloured cytoplasm, low glucose-6-phosphate dehydrogenase(G6PDH) activity) and BCB- group(colourless cytoplasm, high G6PDH activity). After in vitro maturation(IVM), oocytes were subjected to nuclear transfer. SCNT embryos derived from BCB+ oocytes without TSA treatment were used as control, SCNT embryos derived from BCB+ oocytes were treated with 50 nmol/L TSA for 24 h. The result showed that BCB+ oocytes had a significantly greater diameter than that of BCB- oocytes((119.2±1.4) μm vs (106.1±3.0) μm). This result indicated that BCB+ oocytes might have a bigger volume and contain more mRNA and protein needed for subsequent development. Oocyte diameter or oocyte volume has been shown to be associated with G6PDH activity. Timing of the first zygotic cleavage in SCNT embryos derived from BCB+ oocytes was earlier than that of BCB- oocytes and control oocytes. In our earlier study, selecting porcine oocytes for SCNT based solely on morphological criteria such as compaction of cumulus-corona investment and homogeneity of the ooplasm, it was possible to obtain blastocyst production rates of approximately (10.4±1.1)%. However, the results were showed better in the present study which attributed to the use of the BCB test to more competent oocytes. Namely, SCNT embryos derived from BCB+ oocytes achieved significantly higher blastocyst formation rate than other groups((20.0±1.4)% vs (6.6±0.9)% and (10.4±1.1)%). In addition, 50 nmol/L TSA could improve the blastocyst formation rate of reconstructed embryos up to (29.6±2.8)% and some blastocysts espanded and hatched with no blastocysts hatched in control group. SCNT embryos detrived from BCB+ oocytes might have higher developmental competent and 50 nmol/L TSA could improve the development of SCNT embryos. The result suggested that BCB staining selection and TSA treatment may improve the efficiency of SCNT in Sus scrofa ussuricus and provide a way for protecting Sus scrofa ussuricuss germplasm resources

The mature adipocyte dedifferentiation is able to provide homogeneous preadipocyees for researching adpocyte differentiation. In this study, mature adipocytes were isolated and cultured from adipose tissue, and then dedifferentiated to achieve preadipocytes. The subcutaneous adipose tissue from 3-day-old piglets(Sus scrofa) were digested by collagenase type Ⅱ, followed by centrifugation at different centrifugal force and then cultured by "ceiling culture" method. Morphological changes of adipocytes were observed under microscope, and the degree of adipogenesis and differentiation was assessed via oil red O staining. By inducing, adipose-derived progeny cells were redifferentiated into lipid-laden cells with accumulation of lipids. In terms of expression level, peroxisome proliferator-activated receptor-γ (PPARγ) and fatty acid binding protein 4 (FABP4) were detected by Real-time PCR. As we expected, the adipogenic markers, PPARγ and FABP4, increased along with the redifferentiation process. Particularly, a 2.8-fold increase of PPARγ expression, and a 62-fold of FABP4 were shown in the late phase of the redifferentiation process, demonstrating the significantly higher expression levels comparing with the unredifferentiated cells at 0 d(P<0.05). Showing that the predipocytes obtained by dedifferentiation might effectively differentiate to mature adipocytes with adipogenic induction agent. In addition, our study optimized the mature adipocytes culture system as well. Using this system, mature adipocytes can revert into proliferative-competent progeny cells by inducing redifferentiate again into mature adipocytes, which contributes an in vitro model for adipocytes investigation

microRNAs (miRNAs), a class of small ~22-nucleotide-long noncoding RNAs, emerge as key post-transcriptional regulators of gene expression in eukaryotic organisms and involve in a variety of cellular processes, such as cell proliferation, cell differentiation and apoptosis. In silico analysis of the tumor necrosis factor-α (TNF-α) and miR-369 showed that the 3'UTR region of porcine(Sus scrofa) TNF-α adenylate/uridylate-rich elements(AREs) contained two putative miR-369 target sites. The porcine TNF-α 3'UTR region was constructed into the dual-luciferase reporter vector, transfected in the porcine iliac artery endothelial cell (PIEC cell) line to measure the luciferase activity by dual-luciferase reporter assay. The results indicated that the luciferase activity of miR-369 mimics group was significantly lower than that of the control group (P<0.05). Nonetheless, there was not a significant differences between the inhibitors and the control groups (P=0.752). Our results demonstrated that the TNF-α should be the target gene of miR-369 and provides evidence for further research of post-transcriptional mechanisms of TNF-α which affect fat deposition

In order to confirm genetic stability and early fattening expression level difference of foreign porcine growth hormone gene(pGH) in F1 generation of transgenic pigs, growth hormone in 7 trans-pGH gene pigs and 9 non-transgenic pigs of 3-month-old were detected by the advantage of SYBR GreenⅠReal-time PCR method, which were produced by F0 transgenic sows made by nano-gene carrier method induced by sperm and normal boar. According to amplification curve and melting curve maps, Ct mean value was gained through the equation F=(1+E)-ΔΔCt. The results showed that pGH average expression level in F1 generation of transgenic pigs was 1.77 times as non-transgenic pigs. Suggesting that: 1) foreign gene pGH could stably passage in transgenic pigs, 2) not every pGH gene's expression level in transgenic pig was higher than that of non-transgenic pig, 3) average expression level of pGH gene in F1 generation transgenic pigs was observably higher than that of non-transgenic pigs in the same period. A set of molecule biology method of evaluating pGH relative expression level stably was built, and provided scientific method for discussing physical signs and quantitative expression between transgenic and non-transgenic animals

Nuclear enriched abundant transcript 1(NEAT1) is a long non-coding RNA that is involved in immune response, paraspeckle formation and mRNA export. In this study, a single nucleotide polymorphism C1149G in porcine(Sus scrofa) NEAT1 was detected by PCR-RFLP. Allele frequencies varied greatly among different pig breeds examined, and the association results indicated that piglets with the genotype CC had significantly higher levels of Classical swine fever virus (CSFV) antibody (0 and 17 days), mean corpuscular volume(0 days) and mean corpuscular hemoglobin content (32 days), but lower platelet distribution width (32 days), mean platelet volume (32 days) and platelet larger cell ratio (32 days) than those with genotype GG or CG in a Landrace population. The results can be provided as references to molecular markers assisted breeding for pigs

slc30A family(vertebrate cation diffusion facilitator family proteins) transport zinc from the cytoplasm to the lumen, thus decreasing the content of zinc in cytosol. Cloning primers were designed according to the cloning principle of homologous sequence in this paper, the slc30A1~slc30A9 genes were cloned by RT-PCR, then compared cloning sequences of slc30A1~slc30A9 and protein amino acid coding by these gene sequences with other species for homology analysis, and we also studied the slc30A1~slc30A9 distribution in the pig tissue. The main results showed that: The sequence length of gene slc30A1~slc30A9 was about 1 044~2 301 bp all with a complete open reading frame (ORF), which medicated to encode 348~767 amino acid residues, respectively(GenBank accession No. FJ374262.1, EU825192.2, FJ358706.1, EU835903.1, FJ230771.1, FJ237622.1, FJ237623.1, FJ588029 and FJ164069.1, respectively), but slc30A8 was the only mutant. The homology analysis results of slc30A and ZnT(zinc transporter) protein amino acid nearly were the same, higher with human(Homo taurus) and cow(Bos taurus), but lower with Rat(Rattus nurvegicus) and Mouse(Mus musculus), and difference were present in the different isomers and its coding proteins. The results of tissue distribution suggested that slc30A8 was only found in the ileum, slc30A9 was found in the esophagus and all tract, while slc30A1 and slc30A6 were found in other tissue except in the ileum. The other isomers were not found in the ileum, but there were different degree distribution in other tissue. These results suggested that the difference of the slc30A1~ slc30A9 genes distribution in all tissues maybe related to its special function in pig tissue

Aquaporins(AQPs) play importment roles in the water absorbing through gastrointestinal tract as channel proteins. The encoding sequences of aquaporins(AQP2~AQP11) were cloned from the gastrointestinal tract tissue of piglets(Sus scrofa), and then ran bioinformatics analysis. The GenBank accession numbers were EU636238, EU161094, EU165525, EU192130, EU620575, EU024116, EU220426, EU220427, EU582021 and EU220425, respectively. Eight piglets were divided randomly to control and weaning group. Mucosal tissue was collected from the gastrointestinal tract (stomach, duodenum, jejunum, ileum, colon and rectum) and Real-time PCR was applied to determine the mRNA expression of AQPs genes (AQP1, AQP3, AQP8 and AQP11). Bioinformatics software analysis showed that the AQPs had a conserved amino acid sequence of asparagine-proline-alanine(NPA) motif in the protein family. The gene expression results indicated that compared with the control group, mRNA expression of AQP1 was significantly up-regulated in the duodenum, ileum and colon tissue(P<0.05); mRNA expression of AQP3 was significantly up-regulated in the stomach and small intestine tissue(P<0.05); mRNA expression of AQP8 had no significantly difference in duodenum, jejunum, colon and rectum tissue, but up-regulated in the stomach and ileum tissue(P<0.01); The mRNA expression level of AQP11 was increased 1.7 fold in the jejunum tissue. These results suggested the AQPs gene may play a importment role in the regulation of diarrhea in the stomach and small intestines tissues of piglets during weaning-stress

Currently used pTet-on-Advanced regulatory system contains two plasmids, and when it is applied to transgenic animals, two types of genetically modified animals are required to be established and hybridized. This results are in high cost and low efficiency. To address this question, this study aims to construct and functionally characterize tetracycline-inducible single plasmid system. The rtTA and EGFP genes were amplified by PCR from plasmid pTet-on-Advanced and pEGFP-N1, and then sub-cloned into the pTRE-Tight plasmid. The pTRE-Tight-rtTA-EGFP was transfected to fetal porcine fibroblast using liposome transfection.The expression of EGFP under different concentration of doxycyline(Dox)(0.0001, 0.001, 0.01, 0.1 and 1 g/L) were assayed by fluorescence inverted microscope, as the results displayed at 48 h after transfection, there were no expressed green fluorescent in the cells before Dox added, and there were still no expressed green fluorescent in the cells under the concentration of Dox were 0.0001, 0.001, 0.01 and 0.1 g/L until the concentration reached 1 g/L. This phenomenon revealed that the regulation effect of Dox was stimulated with the concentration of 1 g/L. The results showed that the recombinant vector pTRE-Tight-rtTA-EGFP which induced with Tet-on was successfully constructed, and its expression could be induced in fetal porcine fibroblast by Dox. This study provides a novel tool for quantitative transgene expression, and also produces important experimental data to prepare transgenic animals under controlled expression

microRNA has an important regulatory role in cell proliferation and differentiation. The microRNA-378-1 precursor was amplified by PCR from Large White(Sus scrofa) genomic DNA and was digested by XhoⅠ and then ligated into XhoⅠ digested vector pEGP-miR to produce the recombinant vector pEGP-miR-378-1. This vector was transfected to porcine fetal fibroblasts cells (PEF, porcine embryo fibroblast) and its transfection efficiency and expression were detected by fluorescence observation and RT-PCR, respectively. The results showed that pEGP-miR-378-1 vector was constructed successfully with high transfection efficiency. Further RT-PCR results showed that the expression of microRNA-378-1 was increased significantly compared with control groups(P<0.01). This will provide technological platform for producing transgenic animals to further study the function of microRNA-378-1

α-lactalbumin(α-LA) is an important protein in mammalian milk, and contains body essential amino acids and branched-chain amino acids. For its excellent amino acids ratio, easy absorption and functional specificity, α-LA has a great significance for the infant individuals' growth. In this study we aimed to construct human α-LA eukaryotic expression vector pIRES2-ZsGreen1-opLA, and transfected the human α-LA gene into pig(Sus scrofa) fibroblasts to get cells of stable expression of human α-LA. We optimized and synthesized human α-LA mRNA sequence according to the codon preference of pig α-LA gene, and cloned the sequence into the eukaryotic expression vector pIRES2-ZsGreen1, and then proved the correction of the recombinant vector by restriction enzyme digestion and sequencing. The plasmid was transfected into pig fibroblasts cultured by liposome-mediated method, and the cells were divided into three groups: Negative control of transfection group, positive control of empty plasmid transfection group and recombinant plasmid transfection group. The effect of transfection was detected by florescence observation, and the stably transfected cells were selected and cultured by G418, and the target gene expression was detected by RT-PCR. The results of restriction enzyme digestion and sequencing showed that the recombinant plasmid pIRES2-ZsGreen1-opLA was constructed successfully. Observed by fluorescence microscopy, cells of the empty plasmid pIRES2-ZsGreen1 transfection group and the recombinant plasmid pIRES2-ZsGreen1-opLA transfection group both emitted green fluorescence, and the fluorescence was concentrated in the nucleus. The minimum concentration of G418 for screening the recombinant cells was 400 ng/μL. There was an objective gene fragment of the recombinant plasmid transfection cells after RT-PCR, on the contrary, the other two groups cells were not detected such fragments. The eukaryotic expression vector pIRES2-ZsGreen1-opLA has been successfully constructed, and the human α-LA gene can be steadily expressed in pig fibroblasts. These fibroblasts act as the donor cells for the further study of transgenic cloned pigs

The high-density single nucleotide polymorphism(SNP) genotyping platform allows genome wide association studies (GWAS) in pigs. In this GWAS, 355 pigs from a Large White(Sus scrofa)×Minzhu(S. scrofa) intercross population were genotyped using the Illumina Porcine SNP 60K BeadChip, and phenotyped for lean meat weight (LMW) after slaughtered at age of (240±7) d. Association tests between the trait and the SNPs were performed via GWAS using the mixed model and Regression-Genomic Control (GRAMMAR-GC) approach. This GWAS revealed 14 SNPs which showed chromosome-wide significant (P<9.63e-06, SSC1; P<2.37e-05, SSC2; P<1.56e-05, SSC14) association with LWM. On SSC1, two SNPs ALGA0010777 and ALGA0010788, which were located at 285 030 256 and 285 276 856 bp, showed significant association with LWM. Ten significant SNPs were mapped to distal end of SSC2, where the known major gene IGF2 for muscle mass QTL was located. The remainder two SNPs ASGA0065444 and ASGA0065455, which were located at 99 627 980 and 100 078 535 bp on SSC14, were associated with the LWM significantly. The present GWAS results revealed SNPs and candidate genes for LWM in pigs, and the molecular mechanism of LWM difference between Chinese indigenous pig breed and Western commercial pig breed is initially clarified

To investigate the pattern of genetic variation of inbred line Ⅰ of Wuzhishan mini-pig(WZSP)(Sus scrofa) from the 19th to the 21th generations, fifty-three individuals of the inbred line Ⅰand 14 microsatellite loci were used to estimate their genetic heterozygosity(H), polymorphic information content (PIC) and number alleles(N). The results showed that there were a total of 28 alleles at the 14 loci, and the number of allele on F19, F20 and F21 groups were 28, 26 and 24, respectively; average loci alleles were 2.00, 1.86 and 1.70, respectively. In addition，the average PIC and H were 0.24 and 0.31, respectively, which were lower than that of F18 generation; among them, five loci had reached the fully homozygous at the 19th generation as well as did Sw1377 at the 21th generation. But there were a few loci high heterozygosity(H), such as Sw902, S0036 and Sw874. It was speculated that these genes may be related to maintain its a trait in the WZSP. The results of this study are used to provide the basis on inbred line nurture in large animal

Experimental animals are important research content of the research conditions. In past more than 20 years, the Wuzhishan mini-pig(Sus scrofa)(WZSP) inbred line has been carried out by Institute of Animal Science, Chinese Academy of Agricultural Sciences, with inbreeding heddle measure. Up to now, inbreeding F22 of pig's herd have been already obtained which has been applied on human medicine industry. Recently, the whole-genomesequencing of the inbred's WZSP was performed which would be inbred for medical experiments. Compared with human(Homo sapiens), macaque(Macaque mulatta) and rat(Rattus norvegicus), the genome of the inbred pig represented the high homozygosity and specific molecular genetic characteristics. In addition, results from the genome sequencing not only reveal reliability of the innovation in nurturing inbred for Chinese genetic resources, also imply that the inbred line pig is an ideal model in human medical research. The cultivation of Chinese miniature pigs has important practical and historical significance