Transcript variants and expression profiles analysis of Mitf gene in minipigs

Object： To identify transcript variants and expression patterns of porcine Mitf. Materials and methods： A pairwise BLAST search at NCBI database was performed to deduce the structure of porcine Mitf gene. Subsequently, 5＇ RACE and fluorescent quantitative RT-PCR were used to analyze the expression pattern of porcine Mitf in different tissues. Results： Four transcript variants of porcine Mitf, MITF-A, MITF-H, MITF-M and MITF-SUS were identified, all sharing high homology with those in humans, except Mitf-SUS.Conclusion： The sequence of porcine Mitf appear highly homologous to human MITF. However, only 4 transcript variants of porcine Mitf were identified in these minipigs, less than the 9 transcript variants in human MITF.

In vivo Digestive Stability of Soybean β-conglycinin β-subunit in WZS Minipigs

By now, the digestive stability experiments provided by most authoritative organizations are in vitro tests. Evaluating the protein digestive stability with in vivo models should be more objective. The present study aimed to verify the in vivo digestibility of soybean β-conglycinin β-subunit in Wuzhishan （WZS） minipigs. Three minipigs were surgically fitted with O-stomach and T-ileum cannulae and fed with soybean meals. According to SDS-PAGE, the 50 kD fraction of soybean β-conglycinin β-subunit persisted in the gastric fluid until 6 h after feeding, which was detected at 3 h and clearly visible at 4-6 h in the intestinal fluid. Western blot with anti-β-conglycinin β-subunit McAb confirmed it.

Conceptual Proposal of a Facility Design for Accommodating Minipigs for Pharmaceutical and Radiopharmaceutical Research

The use of Animal House Facilities of IPEN-CNEN/SP （Nuclear and Energetic Research Institute-National Nuclear Energy Commission/SP, Brazil） has provided rats and mice with controlled sanitary quality for research and quality control in the production of radiopharmaceuticals lots, produced at IPEN, following rules of CONCEA-Brazilian National Council for Animal Experimentation Control and approved in CEUA （Ethics Committee on the Use of Animals）, before they are sent to hospitals and clinics spread out in Brazil, for use in nuclear medicine. The production and the supply of high quality laboratory animals have fundamental importance for the accomplishment of vanguard scientific research, with reproducibility and universality. The quality of those animals depends, largely, on the available facilities for their production and lodging, to assure the demanded sanitary control and animal＇s welfare, in agreement with the ethical principles that control the activity. Therefore, the facilities design is of vital importance so that the mentioned requirements can be reached. Nevertheless, pigs and miniature pigs have gained importance as large animal models in medicine. With their size, organ capacity, and physiology resembling in several aspects that of humans, pigs are well suited for preclinical experiments and long-term safety studies. Minipigs will be used for preclinical testing of radiopharmaceuticals and assays radioactive materials for cardiac tests. This paper describes the premises and preliminary activities that have been performed at IPEN for the design of new Animal House Facilities dedicated to keeping minipigs taking into account fundamental aspects such as： animal＇s welfare, sanitation, genetic, in agreement with the ethical principles that control the activity and environmental concerns.

Effects of partial portal vein arterialization on liver regeneration after hepatectomy in minipigs with obstructive jaundice

Background Hilar cholangiocarcinoma is a malignant tumor that is difficult to cure. The aim of this study was to observe the effects of flow-controlled partial portal vein arterializations （PPVA） on liver regeneration after hepatectomy in minipigs with chronic obstructive jaundice. Methods Eight minipigs were made into chronic obstructive jaundice models. United semi-hepatectomy, which imitates extended radical surgery for treatment of hilar cholangiocarcinoma, was then performed. The eight minipigs were randomly divided into groups A and B （n=4 minipigs each）. PPVA was performed in Group A but not in Group B. The effects of flow-controlled PPVA on live regeneration after hepatectomy were observed for 30 days after hepatectomy. Results The portal vein PO2 at the immediate time point and on postoperative day 30 was higher in Group A （（47.33±2.43） and （48.50±4.44） mmHg） than in Group B （（35.38±4.06） and （35.55±2.55） mmHg respectively, all P 〈0.01）. The mitotic index of liver cells on postoperative days 14 and 21 was higher in Group A （12.55%±2.85% and 15.25%±1.99% respectively） than in Group B （6.85%±2.10% and 11.88%±1.15% respectively, all P 〈0.05）. The regeneration rate of residual liver on postoperative days 14 and 21 was higher in Group A （24.56%±6.15% and 70.63%±9.83% respectively） than in Group B （11.96%±5.43% and 44.92%±7.42% respectively, P 〈0.05 and P 〈0.01 respectively）.Conclusion Flow-controlled PPVA can promote liver regeneration after hepatectomy and prevent liver failure in minipigs with chronic obstructive jaundice.

Using Huntingtin Knock-In Minipigs to Fill the Gap Between Mouse Models and Patients with Huntington's Disease

Huntington＇s disease （HD） is an inherited autosomal dominant neurodegenerative disease characterized by pro- gressive motor deficits, cognitive decline, and psychiatric symptoms. It is caused by a pathological expansion of CAG trinucleotide repeats in exon 1 of the HD gene, resulting in the translation of a mutant form of huntingtin protein （mutant Htt） with an expanded polyglutamine domain in the N-terminal region [1 ]. Despite great progress in understanding the pathogenesis of HD using multiple mouse models, the exact mechanisms by which mutant Htt induces neuronal dysfunction and death are still not completely clear, and there is no curative treatment for this disease. An important reason is that the mouse, which is the most widely used animal model in HD research, differs from the human in many aspects, including the physiology, drug metabolism, blood-brain barrier, life span, brain volume, and neuroanatomical organization [2]. Thus, it is necessary to establish HD models with higher species than rodents, such as the dog, pig, and non- human primate, so as to bridge the gap between preclinical mouse models and clinical studies.

Analysis of SNPs in the Promoter Region of the Growth Hormone(GH) Gene in Mini-pigs

Single nucleotide polymorphisms(SNPs) of the growth hormone(GH) gene were investigated in six pig breeds,consisting of four mini-pig breeds(Wuzhishan,Bama,Xiang and Tibet pig),and two others(Dahlan and Landrace pig).Three pairs of primers for promoter regions of the GH gene were designed on the basis of the pig genomic sequence and SNPs were detected by the PCR-SSCP method.The results indicated three mutations in the 5’-flanking region.The analysis results showed that the frequencies of allele A and D in four mini-pig breeds were higher than that in other breeds at a locus within the 5’-flanking region(P【0.05).These results suggest that differences in body size may be associated with these SNPs of 5’-flanking region and amino acid mutation of the signal peptide of GH in these pig breeds.

Pathological Changes of Acute Immune Hepatitis in Mini Pigs

Thepurposeofthisstudywastoestablishanexperimentalmodelofa-cuteimmunehepatitiswithpathologicalfeaturessimilartothoseseninhumanacutevi-ralhepatitisfortheuseofexploringitspathogenesisandevaluatingitstherapy.Thehepatocytewaspreparedfromaminipigandusedasantigenandantiserumwasob-tainedfromthegoatbyinjectionofthepreparedantigen.Theacuteimmunehepatitismodelwasestablishedbyinjectingantiserum(2.5ml/kg)intominipigsviathemesen-tericvein.Specimensoftheminipigliversweretakenatdifferentintervalsafteranti-seruminjectionandpathologicfeatureswereobservedbygros,lightandelectronmi-croscopicexaminations.Thecontrolgroupandcarbontetrachloridegroupwerealsoin-cluded.Theresultsshowed:①incontrolgroupthemorphologywasnormal;②incarbontetrachloridegroup,lesionsweremainlylocatedinthecenteroflobule;③thepatholog-icalfeaturesofthisacuteimmunehepatitiscoincidedwiththoseofhumanacuteviralhepatitis.Itissuggestedthatthisexperimentalmodelwouldbeadoptedforotherrele-vantstudies.

Local icariin application enhanced periodontal tissue regeneration and relieved local inflammation in a minipig model of periodontitis

Periodontitis is an inflammatory autoimmune disease. Treatment should alleviate inflammation, regulate the immune reaction and promote periodontal tissue regeneration. Icariin is the main active ingredient of Epimedii Folium, and it is a promising compound for the enhancement of mesenchymal stem cell function, promotion of bone formation, inhibition of bone resorption, alleviation of inflammation and regulation of immunity. The study investigated the effect of icariin on periodontal tissue regeneration in a minipig model of periodontitis. The minipig model of periodontitis was established. Icariin was injected locally. The periodontal clinical assessment index, a computed tomography(CT) scan, histopathology and enzyme-linked immune sorbent assay(ELISA)were used to evaluate the effects of icariin. Quantitative analysis results 12 weeks post-injection demonstrated that probing depth,gingival recession, attachment loss and alveolar bone regeneration values were(3.72 ± 1.18) mm vs.(6.56 ± 1.47) mm,(1.67 ± 0.59)mm vs.(2.38 ± 0.61) mm,(5.56 ± 1.29) mm vs.(8.61 ± 1.72) mm, and(25.65 ± 5.13) mm3 vs.(9.48 ± 1.78) mm3 in the icariin group and0.9% NaCl group, respectively. The clinical assessment, CT scan, and histopathology results demonstrated significant enhancement of periodontal tissue regeneration in the icariin group compared to the 0.9% NaCl group. The ELISA results suggested that the concentration of interleukin-1 beta(IL-1β) in the icariin group was downregulated compared to the 0.9% NaCl group, which indicates that local injection of icariin relieved local inflammation in a minipig model of periodontitis. Local injection of icariin promoted periodontal tissue regeneration and exerted anti-inflammatory and immunomodulatory function. These results support the application of icariin for the clinical treatment of periodontitis.

ENDOCOCHLEAR POTENTIAL AND POTASSIUM CONCENTRATION RECORDING IN MINIPIG COCHLEA

Objective To record the endocochlear potential (EP) and calculate potassium concentration [K+] in Minipig cochleae. Methods We used multi-barreled electrodes to measure the EP and the potential, [K + ]. EP and potassium electrode recording were made in 9 cochleae from 5 minipigs to get normal EP values. Results The average EP value in the cochlea from the minipigs was 77.3 ± 14 mV. The average [K+] for the minipigs was 147.1 ± 13 mM. Conclusions The EP and potential, [K + ] in minipigs are lower than in the human and rodents. This may be the reason why porcine ABR thresholds are slightly higher than humans and rodents.

Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line

Background Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore we investigated the liver expression profile of a highly inbred minipig line. Methods A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme. Results Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5＇ terminal sequences and 140 complete 3＇ terminal sequences.Conclusion These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.