Dequan Xu

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

Background MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood. Methodology We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR. Conclusions Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.

Differential proteome analysis of porcine skeletal muscles between Meishan and Large White

Western and indigenous Chinese pig breeds show obvious differences in muscle growth and meat quality; however, the underlying molecular mechanism remains unclear. In this study, proteome analysis of LM between purebred Meishan and Large White pigs was performed by 2-dimensional gel electrophoresis and mass spectrometry. A total of 25 protein spots were differentially expressed in the 2 breeds. The 14 identified proteins could be divided into 4 groups: energy metabolism, defense and stress, myofibrillar filaments, and other unclassified proteins. Quantitative real-time PCR was used to analyze the partly differentially expressed proteins in mRNA level, which revealed a positive correlation between the content of the proteins and their mRNA levels. We also analyzed the mRNA levels of myosin heavy chain isoforms using quantitative real-time PCR. The results indicated that IIa and IIx fibers were elevated in Meishan pigs, whereas the IIb fiber was more highly expressed in Large White pigs. To the best of our knowledge, this was the first proteomics-based investigation of total skeletal muscle protein in different pig breeds, and these results may provide valuable information for understanding the molecular mechanism responsible for breed-specific differences in growth performance and meat quality.

Selection of reference genes for gene expression studies in porcine skeletal muscle using SYBR green qPCR.

Quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA. Internal controls such as reference genes are used to normalize mRNA levels between different samples for an exact comparison of gene transcription level. However, the expression levels of these reference genes may vary between cell types, developmental stages, species and experimental conditions, thus proper normalization strategy is an important precondition for reliable conclusions. In this study, we explored 10 commonly used reference genes in porcine skeletal muscle using SYBR green qPCR. We used both geNorm and NormFinder to analyze the expression stability and found that PPIA, HPRT and eEF-1γ were suitable internal controls for porcine skeletal muscle. However, PPIA, HPRT and SDHA were suitable for skeletal muscle of western pigs while PPIA, eEF-1γ and HPRT for indigenous Chinese pigs. Normalized qPCR data of ROCK2 were compared with microarray data to evaluate our developed set of reference genes.

Differential proteome and transcriptome analysis of porcine skeletal muscle during development.

To gain further insight into the molecular mechanism of porcine skeletal muscle development, we combined MS characterization of proteins with high-throughput screening of differential mRNAs obtained from purebred Meishan longissimus dorsi muscle (LM) at four stages of 65 days post conception, 3, 60 and 120 days after birth. Strikingly, the dramatic differences were observed in embryo and newborn pigs, whereas 60 and 120 days pigs exhibited similar patterns in protein and mRNA expression. At the protein level, 66 differentially expressed proteins were identified. The development-dependent alterations in protein abundance indicated dramatic changes in metabolism, myofibrillar filaments, cytoskeleton, contractile activity and stress response, and signal transduction. At the transcript level, gene expression was measured with the Affymetrix Porcine Genechip, and 338 genes, representing approximately 1.7% of the chromosome, differed by two fold or more between the neighboring growth phases. Analysis of one such comparison, the expression patterns of most differential proteins showed a positive correlation with their gene expression at the transcript level during skeletal muscle development. Overall, many proteins or genes were previously unrecognized as differentially expressed during growth stages, and they represented novel starting points for understanding the developmental characteristics of biochemical and physiological properties in porcine skeletal muscle.

Molecular characterization and SNPs analysis of the porcine Deleted in AZoospermia Like (pDAZL) gene.

The Deleted in AZoospermia Like (DAZL) gene is expressed in prenatal and postnatal germ cells. In this study, we cloned and characterized the porcine Deleted in AZoospermia Like (pDAZL) gene. We found the full-length coding sequence of the pDAZL encoded a protein of 295 amino acids with a RNA recognition motif (amino acids 41-111) and a DAZ repeat (amino acids 167-120). The deduced protein sequence of pDAZL is 92.5% and 91.5% similar to those of human and bovine, respectively. PCR-MspI-RFLP and PCR-TaqI-RFLP were established to detect an A/G mutation in intron 7 and a C/A mutation in intron 9, respectively. Associations of two SNPs with litter size traits were assessed in Large White (n=275) and DIV (n=128) pig populations, and the statistical analysis demonstrated that CC produced 0.716 more (P<0.05) piglets born alive than CD genotypes in Large White pigs at TaqI locus (C/A mutation in intron 9), and the dominance effect was 0.304 pig per litter (P<0.05). This result suggests that the pDAZL gene might be a good candidate gene of litter size trait and provides some marker information for marker-assisted selection (MAS).

Detection of quantitative trait loci associated with several internal organ traits and teat number trait in a pig population.

Quantitative trait loci (QTL) were detected for 8 internal organ traits, 3 carcass length traits, and teat number trait in 214 pigs in a resource population that included 180 F(2) individuals. A total of 39 microsatellite markers were examined on SSC4, SSC6, SSC7, SSC8, and SSC13. The genetic traits included heart weight (HW), lung weight (LW), liver and gallbladder weight (LGW), spleen weight (SPW), stomach weight (STW), small intestine weight (SIW), large intestine weight (LIW), kidney weight (KW), carcass length to the first cervical vertebra (CL1), carcass length to the first thoracic vertebra (CL2), rib numbers (RNS), and teat numbers (TNS). Results indicated that, 3 highly significant QTL (P <or= 0.01 at chromosome-wise level) for HW (at 30 cM on SSC6), RNS (at 115 cM on SSC7), TNS (at 110 cM on SSC7), and 6 significant QTL (P <or= 0.05 at chromosome-wise level) for LW (at 119 cM on SSC13), LGW (at 94 cM on SSC6), SPW (at 106 cM on SSC8), SIW (0 cM on SSC4), LIW (170 cM on SSC 4), and TNS (at 95 cM on SSC6) were detected. The phenotypic variances for which these QTL were accounted ranged from 0.04 % to 14.06 %. Most of these QTL had not been previously reported.

The formation of brown adipose tissue induced by transgenic over-expression of PPARγ2.

Brown adipose tissue (BAT) is specialized to dissipate energy as heat, therefore reducing fat deposition and counteracting obesity. Brown adipocytes arise from myoblastic progenitors during embryonic development by the action of transcription regulator PRDM16 binding to PPARγ, which promotes BAT-like phenotype in white adipose tissue. To investigate the capability of converting white adipose tissue to BAT or browning by PPARγ in vivo, we generated transgenic mice with over-expressed PPARγ2. The transgenic mice showed strong brown fat features in subcutaneous fat in morphology and histology. To provide molecular evidences on browning characteristics of the adipose tissue, we employed quantitative real-time PCR to determine BAT-specific gene expressions. The transgenic mice had remarkably elevated mRNA level of UCP1, Elovl3, PGC1α and Cebpα in subcutaneous fat. Compared with wild-type mice, UCP1 protein levels were increased significantly in transgenic mice. ATP concentration was slightly decreased in the subcutaneous fat of transgenic mice. Western blotting analysis also confirmed that phosphorylated AMPK and ACC proteins were significantly (P<0.01) increased in the transgenic mice. Therefore, this study demonstrated that over-expression of PPARγ2 in skeletal muscle can promote conversion of subcutaneous fat to brown fat formation, which can have beneficial effects on increasing energy metabolisms and combating obesity.

Alternative splicing of the porcine glycogen synthase kinase 3β (GSK-3β) gene with differential expression patterns and regulatory functions.

Background Glycogen synthase kinase 3 (GSK3α and GSK3β) are serine/threonine kinases involved in numerous cellular processes and diverse diseases including mood disorders, Alzheimer’s disease, diabetes, and cancer. However, in pigs, the information on GSK3 is very limited. Identification and characterization of pig GSK3 are not only important for pig genetic improvement, but also contribute to the understanding and development of porcine models for human disease prevention and treatment. Methodology Five different isoforms of GSK3β were identified in porcine different tissues, in which three isoforms are novel. These isoforms had differential expression patterns in the fetal and adult of the porcine different tissues. The mRNA expression level of GSK3β isoforms was differentially regulated during the course of the insulin treatment, suggesting that different GSK3β isoforms may have different roles in insulin signaling pathway. Moreover, GSK3β5 had a different role on regulating the glycogen synthase activity, phosphorylation and the expression of porcine GYS1 and GYS2 gene compared to other GSK3β isoforms. Conclusions We are the first to report five different isoforms of GSK3β identified from the porcine different tissues. Splice variants of GSK3β exhibit differential activity towards glycogen synthase. These results provide new insight into roles of the GSK3β on regulating glycogen metabolism.

A method for obtaining DNA from compost

An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical–chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme HhaI.

Multiple alternative splicing and differential expression of actinin-associated LIM protein (ALP) during porcine skeletal muscle development in vitro and in vivo

The actinin-associated LIM protein (ALP) is co-localized with alpha-actinin at the Z-discs and plays a critical role in the integration of cytoskeletal architecture and transcriptional regulation. Here we report that five isoforms of the porcine ALP were generated in skeletal muscle by alternative splicing. All of ALP isoforms were predominantly expressed in skeletal muscle except for ALP2. These isoforms had different expression profiles during the prenatal and postnatal period of the porcine skeletal muscle development and between the two breeds. Moreover, ALP1 and ALP3 were expressed at higher levels in soleus and masseter muscles compared with longissimus dorsi and bicepsfemoris muscles in Yorkshire pigs. Expression analysis in porcine satellite cells showed that all isoforms were induced in differentiated porcine satellite cells, suggesting a role of them in myogenic differentiation. These results provide new insight into roles of regulation at level of splicing of the ALP in governing porcine skeletal muscle development.