Jiaqi Li

Cloning and characterization of the 5′-flanking region of the pig adiponectin gene.

Adiponectin, a cytokine hormone secreted exclusively by adipose tissue, has key roles in energy homeostasis, and in glucose and lipid metabolism. To understand the regulatory expression of pig adiponectin, the 5-flanking region of the adiponectin gene was isolated from a pig BAC library. 5-RACE analysis revealed that there were three transcriptional start sites, including one that is novel. The luciferase reporter assay detected a positive cis-acting element for efficient expression of the adiponectin gene at the region spanned by nucleotides -1150 to -1130 with serially deleted 5-flanking sequences as its promoters. Analysis of oligonucleotide competition by the electrophoretic mobility-shift assay revealed the presence of a cAMP-responsive element (CRE) (nucleotides -1150 to -1130) for the cAMP-responsive element binding protein (CREB), which has not been reported in human or mouse adiponectin genes. These results indicated that CREB is an essential regulatory factor for the transcriptional activity of pig adiponectin.

Overexpression of SIRT6 in Porcine Fetal Fibroblasts Attenuates Cytotoxicity and Premature Senescence Caused by D-Galactose and Tert-Butylhydroperoxide

SIRT6, a member of the yeast silent information regulator 2 (SIR2) family, possesses both robust ADP-ribosyltransferase activity and protein deacetylase activity depending on NAD(+). It has been shown to maintain genomic stability and telomere integrity, and to prevent age-related disorders and premature ageing. However, the mechanism by which SIRT6 overexpression affects cellular ageing is not well understood. In this study, we investigated the effect of SIRT6 overexpression on cytotoxicity and ageing syndromes. A full-length cDNA of porcine SIRT6 was inserted into pcDNA3.1(-) and subsequently transfected into porcine fetal fibroblasts (PFFs). Overexpression of SIRT6 was identified by quantitative real-time polymerase chain reaction and western blot assay. The cells were incubated with D-galactose and tert-butylhydroperoxide at their cytotoxic concentrations. The damage caused by the stresses was detected with fluorescence microscopy using 4,6-diamidino-2-phenylindole (DAPI) staining, DNA ladder analysis, and observation under transmission electron microscopy. The results showed that SIRT6 overexpression in cells decreased damage to the nuclei. It also protected against the generation of DNA fragmentation and damage in the ultramicrostructure of the cells, especially damage to mitochondria. Our observations suggested that one function of SIRT6 in PFFs was to dampen cytotoxicity, and, therefore, to decrease the damage that causes premature senescence.

Selection of reference genes for studies of porcine endometrial gene expression on gestational day 12.

Comparing gene expression patterns in the endometrium on gestational day 12 (GD12) between Erhualian (ER) and Landrace×Large White (LL) pigs is helpful to understand the biological mechanisms of fecundity. Selecting genes that have stable expression levels as the internal standards in a comparative study is essential for identifying real gene-specific variation by quantitative RT-PCR (qRT-PCR). Five genes expressed in sow endometria on GD12 were evaluated for their suitability as internal control for relative quantification by qRT-PCR. These genes were beta-actin (ACTB), beta-2-microglobulin (B2M), phosphoglycerate kinase 1 (PGK1), RNA polymerase II polypeptide G (RPG), and ribosomal protein S20 (RPS20), which represent different functional classes. Our results indicated that ACTB, B2M, and PGK1 were not suitable as internal standards for normalization because of their huge variability between the two breeds. RPS20 and RPG were most stable, and the former is recommended to serve as the internal standard when the use of multiple housekeeping genes is unpractical.

Sequence identification, chromosomal mapping and tissue specific expression of the porcine paraoxonase 1 (PON1) gene

Serum paraoxonase (PON1) plays an important role in protecting low-density lipoprotein against oxidative modification. In this study, the full-length cDNA sequence (1,416xa0bp) of porcine PON1 was cloned by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. It was found to contain a 1,068xa0bp open reading frame encoding a deduced protein of 355 amino acids with a calculated molecular weight of 40.02xa0kDa. The genomic structure and sequence of porcine PON1 were also analyzed using a bacterial artificial chromosome clone of a Chinese Erhualian pig. The porcine PON1 gene contained nine exons and eight introns spanning approximately 29xa0kb, and was located on chromosome nine between microsatellite markers SWR915 and SW944 by IMpRH mapping. Porcine PON1 was highly expressed in kidney, followed by liver, lung and small intestine, expressed at an extremely low level in heart, and was hardly expressed in spleen, lymph, muscle of the anterior limb, cerebrum, fat, cerebellum or hypothalamus.

Identification of novel transcripts from the porcine MYL1 gene and initial characterization of its promoters

The fast skeletal alkali myosin light polypeptide 1 (MYL1) gene is one of three mammalian alkali MLC genes and encodes two isoforms, 1f and 3f, which play a vital role in embryonic, fetal, and adult skeletal muscle development. We isolated the MYL1 gene from a pig BAC library with the goal of characterizing its promoter and identifying its transcripts. Genes and isoforms were identified by reverse transcriptase-PCR, northern blot and RACE (Rapid Amplification of cDNA Ends). Potential MYL1 gene promoters were characterized using a luciferase reporter assay and electrophoretic mobility shift assays (EMSA). MLC1f, MLC3f, and three additional isoforms of porcine MYL1, MLC5f-A, -B, and -C were identified. Up to now, the three novel isoforms had not been reported in human or mouse. Northern blot analysis indicated that MLC1f, MLC3f, and MLC5fs were expressed only in longissimus dorsi muscles. Two transcription initiation and termination sites were identified by RACE. Promoter analysis and EMSA demonstrated the presence of a MEF3 (skeletal muscle-specific transcriptional enhancer) binding site (+384 to +481), which might be essential for porcine MYL1 transcription. Our results suggested that five transcript variants were generated using alternative promoters, two transcription start sites, and polyA sites, as well as variable splicing of the pig MYL1 exon 5. The identification of alternative promoters and splice variants, the expression of the splice variants in different muscle tissues, and the definition of regulatory elements provide important molecular genetic knowledge concerning the MYL1 gene.

Molecular characterization and expression analysis of caveolin-1 in pig tissues.

Members of the caveolin family played important roles during fundamental cellular processes, such as regulation of cell morphology, migration, and gene expression in muscle cells. In this study, caveolin-1 (Cav-1), one of the caveolins, was identified from longissimus dorsi muscle of Large Yorkshire pig and Chinese indigenous Lantang pig based on the results of mRNA differential display analysis. The deduced amino acids sequence of the porcine Cav-1 contained a caveolin domain, and was very conservative among different species. The Cav-1 mRNA was widely expressed in the eight tissues in this study, including heart, liver, kidney, encephalon, spleen, lung, longissimus dorsi muscle, and back fat, and the highest expression quantity was found in back fat of the two pig breeds. The expression quantity of porcine Cav-1 in back fat and longissimus dorsi muscle of Lantang pig was significantly higher than that of Large Yorkshire (P<0.01, and P<0.05, respectively). These results suggested that the Cav-1 might be a candidate gene for carcass traits, and might provide valuable information for understanding the mechanism of caveolae signaling in fat deposition by using the animal model of pig.

Cloning and characterization of the 5'-flanking region of the pig AgRP gene.

Agouti-related peptide (AgRP), a brain neuropeptide generated by AgRP/neuropeptide Y (NPY) neurons, plays a vital role in the hypothalamic regulation of energy homeostasis. RT–PCR and real-time PCR were carried out in various tissues to detect the AgRP expression pattern in pigs. Our RT–PCR results showed that the pig AgRP gene was ubiquitously expressed in all examined tissues including heart, liver, spleen, lung, kidney, stomach, bladder, m. longissimus, belly fat, brain, large intestine, lymph, back fat, skin, and hypothalamus. Real-time quantitative PCR experiments revealed that it is in the hypothalamus with the highest expression of AgRP both in adult Lantang and Landrace pigs compared to the back fat and m.longissimus muscle and the cDNA level of AgRP in the hypothalamus of adult Chinese indigenous Lantang pig (fat-type) is significantly higher than that of Landrace pig (lean-type). To understand the regulation of the pig AgRP gene, the 5′-flanking region was isolated from a pig bacterial artificial chromosome library and used in a luciferase reporter assay. A positive cis-acting element for efficient AgRP expression was identified at nucleotides −501 to −479, by 5′-serial deletion of the promoter. Electrophoretic mobility-shift assays (EMSA) with competing oligonucleotides revealed that the critical region contained a cis-acting element for Neurogenic Differentiation (NeuroD), which is a member of the NeuroD family of basic-helix-loop-helix transcription factors. This element has not been reported in human or mouse AgRP genes. Our results indicated that NeuroD might be an essential regulatory factor for transcription of pig AgRP, providing an important clue about energy homeostasis regulation in the porcine and human brain.

Sequence Identification, Tissue Distribution and Polymorphism of the Porcine Cathepsin D (CTSD) Gene

Cathepsin D (CTSD), a major ubiquitously expressed aspartic protease, is not only involved in muscle protein degradation, but also related to some pathological processes. In this study, we characterized the full-length cDNA, genomic DNA sequence, expression profile and polymorphism of the porcine CTSD gene. The full-length cDNA of porcine CTSD gene and the predicted protein sequence shared high identities wih other mammalian orthologous. Northern-blot analysis and Reverse transcription (RT)-PCR results indicated that the CTSD gene has one transcript of approximately 2.0 kb in normal tissues and was expressed ubiquitously in pigs, without significant differences in porcine heart, liver, spleen, lung, kidney, stomach, fat, triceps brachi, biceps femoris, and longissimus muscles. The porcine CTSD gene spans ∼ 9.0 kb including nine exons. All exon/intron boundaries adhere to the GT/AG rule. Altogether 35 nucleotide polymorphisms of CTSD gene were discovered between Duroc, Landrace, Erhualian, and Dahuabai pigs. These polymorphisms included three missense mutations, eight synonymous mutations, and 24 intronic substitutions, and most polymorphisms are located in the intron 4 and 5. Three polymorphisms were genotyped in Duroc, Landrace, Dahuabai, and Erhualian pigs by PCR-RFLP method, and significant differences of their genotype frequencies were observed between Chinese native breeds (Dahuabai and Erhualian) and western breeds (Duroc and Landrace).

Cloning and Characterization of the 5′-Flanking Region of the Pig Cocaine- and Amphetamine-Regulated Transcript Gene

The cocaine- and amphetamine-regulated transcript (CART) gene encodes an anorexigenic peptide. It has a key role in the hypothalamic regulation of energy balance through reducing food intake and enhancing lipid substrate utilization. To detect the CART expression pattern in pigs, reverse transcription (RT)-polymerase chain reaction (PCR) and real-time PCR were performed in various tissues. Our RT-PCR results revealed that the pig CART gene was ubiquitously expressed in all examined tissues including hypothalamus, m. longissimus, backfat, heart, liver, spleen, lung, kidney, stomach, bladder, belly fat, brain, large intestine, lymph, and skin. Real-time quantitative PCR experiments revealed that the cDNA level of CART in both the hypothalamus and backfat of adult Landrace pig (lean-type) was significantly higher than that of Chinese indigenous Lantang pig (fat-type), and it was in the hypothalamus where the highest expression of CART was observed for both adult Lantang and Landrace pigs, compared with backfat and m. longissimus muscle. To understand the regulation of the pig CART gene, the 5-flanking region was isolated from a pig bacterial artificial chromosome library and used in a luciferase reporter assay. A positive cis-acting element for efficient CART expression was identified at nucleotides -73 to -53, using 5-serial deletion of the promoter. Electrophoretic mobility shift assays with competing oligonucleotides revealed that the critical region contained a cis-acting element for the zinc-binding protein factor, a zinc-finger transcription factor of the Kruppel family. This element has not been reported in human or mouse CART genes. Our results indicated that zinc-binding protein factor might be an essential regulatory factor for transcription of pig CART, providing important insight into mechanisms involved in energy homeostasis regulation in the porcine and human brain.

Molecular characterization, expression profile and polymorphisms of the porcine TNNC2 gene.

Fast skeletal troponin C (TNNC2) plays a key role in the regulation of muscle contraction, and modulates the Ca2+-activation characteristics of muscle fibers. In this study, we characterized the full-length cDNA of the porcine TNNC2 gene, which is composed of a 65 bp 5UTR, a 483 bp (ORF) and a 174 bp 3 UTR. Northern blot analysis indicated that the TNNC2 gene had one transcript of approximately 0.7 kb and was expressed exclusively in skeletal muscle (triceps brachii, biceps femoris and longissimus dorsi). SDS-PAGE and Western blotting analysis indicated that the porcine TNNC2 protein comprised 160 amino acids, and had a molecular mass of 18 kDa. The porcine TNNC2 gene, which spans 3.2 kb, was isolated and was found to be composed of six exons. All exon/intron boundaries adhered to the GT/AG rule. Altogether 20 nucleotide polymorphisms of the TNNC2 gene were discovered in Duroc, Landrace, Lantang and Dahuabai pigs, and included three missense mutations and 17 intronic substitutions. Five of the polymorphisms were genotyped in Lantang, Dahuabai, Landrace, Large White and Duroc pigs using PCR-RFLP. Significant differences in genotype frequencies were observed between the Chinese native breeds (Lantang and Dahuabai) and the western breeds (Landrace, Large White and Duroc). These datas have identified the TNNC2 gene as an excellent model system for studies of developmentally regulated gene expression and a novel marker suitable for studies of the association of candidate genes with growth traits in porcine skeletal muscle.