Haitao Shang

Tissue distribution of CYP3A29 mRNA expression in Bama miniature pig by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)

Pig or minipig cytochrome P450 (CYP) 3A isozymes have been proven to be a good model of human drug metabolism, and human CYP3A seems to be well modelled by pig or minipig CYP3A29. We studied CYP3A29 mRNA expression in ten tissues (liver, duodenum, kidney, adrenal gland, skin, brain, lung, testis, uterus and ovary) in different age groups of Bama miniature pig by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In adult pig (4 and 6 months old), the most relative expression by normalization to β-actin of CYP3A29 mRNAs of tissues is in liver; then in duodenum, skin and kidney. Values of CYP3A29 in the extrahepatic tissues except for duodenum are very low compared with that in liver. In new-borns the mean CYP3A29 expression in duodenum was higher than that in liver, and the expression in skin was very low while high in 40 days, 4 months and 6 months age groups. For different ages, the increased CYP3A29 expression in liver and duodenum was detected with age. The effective CYP3A29 induction in liver was also detected by rifampin oral intubation. Apparent interindividual variability of CYP3A29 mRNA expression in Bama miniature pigs was detected. This work will broaden the understanding of the physiological functions of CYP3A29 in miniature pig, and promote Bama miniature pig’s application in pharmacological and toxicological studies.

Constitutive expression of CYP3A mRNA in Bama miniature pig tissues.

The pig, particularly the miniature pig (minipig), is becoming an important animal model due to its physiological and anatomical similarities to humans. Bama minipigs (Sus scrofa domestica), a Chinese natural minipig breed, are a promising animal model. The pig is a useful model for drug metabolism and pharmacological studies due to the similar properties of cytochrome P450 (CYP)3A between pigs and humans. However, a detailed investigation regarding the abundance and expression of CYP3A in porcine tissues, particularly in minipig tissues, has not been performed. The present study investigated constitutive expression of CYP3A mRNA in Bama minipig tissues using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). We found that the expression of CYP3A mRNAs relative to the endogenous control, β-actin (ACTB), was lower than when compared to the expression of the endogenous control, TATA-binding protein (TBP), except for the expression of CYP3A29 mRNA in the spleen, adrenal gland, testis, and epididymis, and CYP3A46 in the spleen. Expression levels of all three CYP3As were highest in the liver amongst all the tissues tested, and the order of relative mRNA expression level of the three CYP3As was different between other tissues. We also analyzed the relative expression of the three CYP3A mRNAs in each tissue. CYP3A46 had the highest expression in all extrahepatic tissues, whereas CYP3A22 had the highest expression in the liver, and CYP3A29 had the lowest expression in all tissues except in the duodenum, where it had higher expression than CYP3A22. Because CYP3A22 and CYP3A46 were the most highly expressed isoforms, it could follow that they are probably important functional CYP3A genes for Bama miniature pig. Our present work will broaden the understanding of the physiological functions of CYP3As in the Bama minipig and promote its application in drug metabolism and pharmacological studies. Our results also indicate that the breed, age, and castration status of the pig should be considered when using the pig as an animal model in pharmacological applications.

Proteomic analysis of the skin of Chinese giant salamander (Andrias davidianus).

UNLABELLED The Chinese giant salamander (Andrias davidianus), renowned as a living fossil, is the largest and longest-lived amphibian species in the world. Its skin has developed mucous gland which could secrete a large amount of mucus under the scraping and electric stimulation, and the molting is the degraded skin stratum corneum. Although several proteomic studies have focused on functional proteomes of mammalian and frog skin, the skin proteome of Chinese giant salamander has not yet been carefully studied. To establish the functional skin proteome of Chinese giant salamander, two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) were applied to detect the composition and relative abundance of the proteins in the skin, mucus and molting. Our findings indicated that 249 proteins were identified in the skin, 155 proteins in the mucus, and 97 proteins in the molting. Furthermore, Gene Ontology (GO) analysis showed that these proteins participated in various physiological activities, including extracellular matrix organization, defense, immune response, wound healing, respiration, etc. In conclusion, the proteomic results provide new insight in the aspects of the proteomes in the skin, mucus and the molting of Chinese giant salamander. BIOLOGICAL SIGNIFICANCE This was the first study to examine the protein expression abundance in the skin, mucus and molting of Chinese giant salamander by a proteomics approach. Meantime, the identification of a more global proteome in normal skin may provide a basis for characterizing and comparing the skin proteomes from other amphibian species.

Lentiviral Transgenic MicroRNA-Based shRNA Suppressed Mouse Cytochromosome P450 3A (CYP3A) Expression in a Dose-Dependent and Inheritable Manner

Cytochomosome P450 enzymes (CYP) are heme-containing monooxygenases responsible for oxidative metabolism of many exogenous and endogenous compounds including drugs. The species difference of CYP limits the extent to which data obtained from animals can be translated to humans in pharmacodynamics or pharmacokinetics studies. Transgenic expression of human CYP in animals lacking or with largely reduced endogenous CYP counterparts is recognized as an ideal strategy to correct CYP species difference. CYP3A is the most abundant CYP subfamily both in human and mammals. In this study, we designed a microRNA-based shRNA (miR-shRNA) simultaneously targeting four members of mouse CYP3A subfamily (CYP3A11, CYP3A16, CYP3A41 and CYP3A44), and transgenic mice expressing the designed miR-shRNA were generated by lentiviral transgenesis. Results showed that the CYP3A expression level in transgenic mice was markedly reduced compared to that in wild type or unrelated miR-shRNA transgenic mice, and was inversely correlated to the miR-shRNA expression level. The CYP3A expression levels in transgenic offspring of different generations were also remarkably lower compared to those of controls, and moreover the inhibition rate of CYP3A expression remained comparable over generations. The ratio of the targeted CYP3A transcriptional levels was comparable between knockdown and control mice of the same gender as detected by RT-PCR DGGE analysis. These data suggested that transgenic miR-shRNA suppressed CYP3A expression in a dose-dependent and inheritable manner, and transcriptional levels of the targeted CYP3As were suppressed to a similar extent. The observed knockdown efficacy was further confirmed by enzymatic activity analysis, and data showed that CYP3A activities in transgenic mice were markedly reduced compared to those in wild-type or unrelated miR-shRNA transgenic controls (1.11±0.71 vs 5.85±1.74, 5.9±2.4; P<0.01). This work laid down a foundation to further knock down the remaining murine CYP3As or CYPs of other subfamilies, and a basis to generate CYP knockdown animals of other species.

Transcriptome Profiling Identifies Differentially Expressed Genes in Postnatal Developing Pituitary Gland of Miniature Pig

In recent years, Tibetan pig and Bama pig are popularly used as animal models for medical researches. However, little genomic information is available for the two breeds, particularly regarding gene expression pattern at the whole-transcriptome level. In this study, we characterized the pituitary transcriptome profile along their postnatal developmental stages within and between the two breeds in order to illustrate the differential dynamics and functions of differentially expressed genes. We obtained a total of ∼300 million 80-bp paired-end reads, detected 15 715 previously annotated genes. Most of the genes (90.33%) were shared between the two breeds with the main functions in metabolic process. Four hormone genes (GH, PRL, LHB, and FSHB) were detected in all samples with extremely high levels of expression. Functional differences between the three developmental stages (infancy, puberty and adulthood) in each breed were dominantly presented by the gene expressions at the first stage. That is, Bama pig was over-represented in the genes involved in the cellular process, while Tibetan pig was over-represented in the genes represented by the reproductive process. The identified SNPs indicated that the divergence between the miniature pig breeds and the large pig (Duroc) were greater than that between the two miniature pig breeds. This study substantially expands our knowledge concerning the genes transcribed in the pig pituitary gland and provides an overview of pituitary transcriptome dynamics throughout the period of postnatal development.

A reference gene set construction using RNA-seq of multiple tissues of Chinese giant salamander, Andrias davidianus

Abstract Background Chinese giant salamander (CGS) is the largest extant amphibian species in the world. Owing to its evolutionary position and four peculiar phenomenon of life (longevity, starvation tolerance, regenerative ability, and hatch without sunshine), it is an invaluable model species for research. However, lack of genomic resources leads to fewer study progresses in these fields, due to its huge genome of ∼50 GB making it extremely difficult to be assembled. Results We reported the sequenced transcriptome of more than 20 tissues from adult CGS using Illumina Hiseq 2000 technology, and a total of 93 366 no-redundancy transcripts with a mean length of 1326 bp were obtained. We developed for the first time an efficient pipeline to construct a high-quality reference gene set of CGS and obtained 26 135 coding genes. BUSCO and homologous assessment showed that our assembly captured 70.6% of vertebrate universal single-copy orthologs, and this coding gene set had a higher proportion of completeness CDS with comparable quality of the protein sets of Tibetan frog. Conclusions These highest quality data will provide a valuable reference gene set to the subsequent research of CGS. In addition, our strategy of de novo transcriptome assembly and protein identification is applicable to similar studies.

Expression of Bama Minipig and Human CYP3A Enzymes, Comparison of the Catalytic Characteristics with Each Other and Their Liver Microsomes

Minipigs represent a good animal model because of the physiologic and anatomic similarities they share with humans. Three cytochrome P450 (CYP) 3A isozymes, CYP3A22, CYP3A29, and CYP3A46, have recently been reported to be expressed in Bama minipigs, which have limited data relating to their metabolic characteristics. In the present study, Bama minipig CYP3A22, CYP3A29, and CYP3A46 were recombinantly expressed and their metabolic manners were compared with those of human CYP3A4 and CYP3A5 and also human and Bama minipig liver microsomes. The results indicated Bama minipigs and human CYP3A enzymes showed similar metabolic kinetics and metabolite profiles using testosterone, midazolam, and nifedipine as substrates. However, the differences in amino acid sequences change the elimination velocity and metabolic preference of CYP3A enzymes to their substrates. It was demonstrated that CYP3A29, CYP3A4, and CYP3A5 were the most active enzymes for all reactions, whereas CYP3A46 was the least active enzyme. Substrate-dependent metabolism characteristics between human and Bama minipig CYP3A isoenzymes exist.

Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis

Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF+/L247S) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITFL247S/L247S) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

Data from proteomic analysis of the skin of Chinese giant salamander (Andrias davidianus).

The Chinese giant salamander (Andrias davidianus), renowned as a living fossil, is the largest and longest-lived amphibian species in the world. Its skin is rich in collagens, and has developed mucous gland which could secrete a large amount of mucus under the scraping and electric stimulation. The molting is the degraded skin stratum corneum. To establish the functional skin proteome of Chinese giant salamander, two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) were applied to detect the composition and relative abundance of the proteins in the skin, mucus and molting. The determination of the general proteome in the skin can potentially serve as a foundation for future studies characterizing the skin proteomes from diseased salamander to provide molecular and mechanistic insights into various disease states and potential therapeutic interventions. Data presented here are also related to the research article “Proteomic analysis of the skin of Chinese giant salamander (Andrias davidianus)” in the Journal of Proteomics [1].