过表达Sirt2抑制猪前体脂肪细胞的分化

本文旨在利用过表达技术研究Sirt2在猪前体脂肪细胞分化中的作用.首先将Sirt2插入腺病毒穿梭载体pAdTrack-CMV,并与骨架载体pAdEasy-1在大肠杆菌BJ5183中同源重组,重组体用Lipofectamine2000包装转染HEK293细胞系,成功获得重组腺病毒vAd-Sirt2.用vAd-Sirt2感染猪前体脂肪细胞,48h后油红O染色法观察脂肪细胞分化情况,RT-PCR检测脂肪细胞分化标志基因PPARγ和aP2的表达.结果显示,过表达Sirt2促使细胞中脂滴减少,同时标志基因PPARγ、aP2mRNA水平显著降低,说明Sirt2抑制猪前体脂肪细胞分化,这为控制猪体脂沉积提供依据以及为人类肥胖和相关疾病的治疗和预防奠定基础.     

A contemporary atlas of the mouse diaphragm: myogenicity, vascularity, and the pax3 connection

The thoracic diaphragm is a unique skeletal muscle composed of costal, crural, and central tendon domains. Although commonly described in medical textbooks, newer insights into the diaphragm cell composition are scarce. Here, using reporter mice, combined with gene expression analyses of whole tissues and primary cultures, we compared the diaphragm domains and their myogenic progenitors (i.e., Pax3/7 satellite cells). The outcomes of these analyses underscore the similarities between the myogenic aspects of the costal and crural domains. Expression levels of all myogenic genes examined (except Pax3) were strongly affected in mdx (dystrophin-null) mice and accompanied by an increase in fibrosis- and adiposity-related gene expression. Cell culture studies further indicated the presence of a non-myogenic Pax3-expressing population, potentially related to vascular mural cells. We additionally investigated the diaphragm vasculature. XLacZ4 and Sca1-GFP transgenes allowed a fine definition of the arterial and microvasculature network based on reporter expression in mural cells and capillary endothelium, respectively. We also provide insights into the organization of the diaphragm venous system, especially apparent in the central tendon and exhibiting arcades lined with fat-containing cells. The novel information in this "contemporary atlas" can be further explored in the context of diaphragm pathology and genetic disorders.     

Validation and fine mapping of a QTL for ovulation rate on swine chromosome 3

Ovulation rate (OR) is an important component of litter size, but mutation(s) in gene(s) underlying OR QTL have yet to be identified in pigs. Markers within an OR QTL on SSC3 were genotyped in three white composite lines selected for ten generations for increased OR or uterine capacity (UC), with one line being an unselected control. Numbers of corpora lutea (CL) and UC (number of fully formed fetuses) were collected at approximately 105 days of gestation, as well as ovary weight (OW), uterine length (UL) and uterine weight (UW) measurements at 160 d of age in generation 12 and 13 females from all three lines. Six microsatellites and ten single nucleotide polymorphisms (SNPs; 0–42 cM) were genotyped in pigs from all lines of generations 11 through 13. The allele frequencies of 24269.1, SW2429, 7907.2 and 7637.2 were different (P < 0.01) in the OR line compared to the control line. A significant (P < 0.05) association of CL with 24269.1 (additive effect 0.65 ± 0.32) was detected, and additive genotypic effects approached significance for markers at 28 through 35 cM (16963.2, 27514.1 and SWR1637). Haplotyping of 7637.2 and 16963.2 (31 through 32 cM) identified a significant additive association of haplotype 1 with CL (?0.62 ± 0.30). These markers were also associated with OW (24296.1 and SWR1637), UL (16963.2, 27514.1 and haplotypes of 7637.2/16963.2) and UW (haplotypes of 7637.2/16963.2). This study verifies an OR QTL on SSC3. However, based on the data, it was concluded that there may be two genes, at 13 through 18 cM and 28 through 35 cM, controlling OR on SSC3p.     

Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos: Evaluation of critical steps of the procedure

Previous trials achieved extremely poor results when using the one-step warming method in a syringe in combination with non-surgical deep intrauterine transfer (NET) of superfine open pulled straw (SOPS)-vitrified embryos. This study aimed to assess the effect of the warming procedure on the in vitro and in vivo development of SOPS-vitrified embryos. The effect of the passage of the vitrified-warmed (VW) embryos through the NET catheter was also evaluated. Groups of 4 to 6 morulae and blastocysts, collected from weaned sows, were SOPS-vitrified in 1 μL of vitrification medium, warmed by the one-step warming method in a dish or in a 1-mL syringe and cultured in vitro for 48 h to evaluate the embryo survival (ES) and hatching rates (HR). Warming in syringe had a deleterious effect (P < 0.05) on the in vitro ES (60.5 ± 10.4%) and HR (39.6 ± 9.5%) of VW embryos in comparison with embryos warmed in a dish (85.4 ± 10.6% and 69.0 ± 8.4%, respectively). This decreased embryonic development was due to the increased time required between the removal of the straws from the liquid nitrogen and the contact of the embryos with the warming medium when the warming was performed in a syringe in comparison with that for the warming in a dish. After verifying that the passage of VW embryos through the NET catheter does not have a damaging effect on their further in vitro development, the negative effect of warming in a syringe was also confirmed after NET. Fifteen fresh and SOPS-vitrified embryos warmed in a syringe or in a dish were transferred to each recipient (n = 28) and recovered 24 h later to assess their developmental progression. All embryos from the syringe group were found to have degenerated at recovery. The in vivo ES and HR from the dish group (80.4 ± 3.4% and 14.2 ± 7.2%, respectively) were lower (P < 0.05) than those from the fresh group (94.0 ± 4.1% and 36.8 ± 7.8%, respectively). Combining the warming in a dish and the NET procedure, 35 VW embryos were transferred to each of 10 gilts. Five recipients farrowed an average of 10.4 ± 0.9 piglets. In conclusion, the method of one-step warming in a syringe has a negative effect on the in vitro and in vivo viability of SOPS-vitrified porcine embryos. In addition, NET of SOPS-vitrified embryos warmed by the one-step method in a dish showed promising reproductive performance of recipients. However, despite the great potential of this technology, further developments are required for large-scale commercial applications.     

Characterization of the porcine KIT ligand gene: expression analysis, genomic structure, polymorphism detection and association with coat colour traits

Kit ligand (KITLG) is the ligand for the type III receptor tyrosine kinase KIT. Studies of the KIT/KITLG pathway in a number of mammalian species have shown that it is important for the development of stem cell populations in haematopoietic tissues, germ cells in reproductive organs and the embryonic migrating melanoblasts that give rise to melanocytes. Consequently, mutations in the pathway may result in a range of defects including anaemia, sterility and de-pigmentation. The cDNA sequence of the porcine KITLG gene has been reported previously, and is an attractive candidate locus for moderating coat colour in pigs. In this paper we report the gene structure and physical mapping of the porcine gene. We also report the identification of polymorphisms in the gene, one of which was used to confirm linkage to chromosome 5. Preliminary RNA expression studies using a panel of tissues have shown that in addition to the known variant lacking exon 6, there is alternative splicing of exon 4. However, little evidence was found for the KITLG gene being linked to variation in colour in a Meishan x Large White cross.     

Polymorphisms in the promoter region of the porcine antiviral MX1 and MX2 genes

The porcine MX1 and MX2 promoters were characterized in this study. Sequencing of the 332-bp MX1 promoter region identified 15 substitutions and insertions at three positions in 21 pigs from 15 breeds, in which nine genotypes were classified. Among the nine genotypes, no statistically significant differences in the promoter activities were observed after interferon (IFN- α 2b) treatment of transiently transfected cells containing constructs with luciferase reporter plasmids. The 341-bp MX2 promoter region contained regulatory sequences for ISRE, GC box, Sp1 and AP-1, as well as a TATA box. Nucleotide sequences of the MX2 promoter region revealed four substitutions and one deletion, in which six genotypes were classified. Among the six genotypes, a statistically significant difference ( P  < 0.05) in MX2 promoter activities after IFN- α 2b treatment was detected in transiently transfected cells.