Abstract:Objective To knock out the Abcb1 gene of rat,and establish the Abcb1 humanized rat model based on the Abcb1 knock out rat. Methods The animal model was established using BAC and CRISPR/Cas9 technology,and was analyzed by PCR, RT-PCR and real-time PCR. Results Establishing a rat model expressing human Abcb1 stably by transfer the 153 kb BAC containing human Abcb1 promoter and cDNA into rat genome, and establishing the Abcb1 knock out rat at the same time. Establishing the Abcb1 humanized model by crossing these two strains together. The expression pattern of Abcb1 in Abcb1 humanized rat is different from the wild type rat. The Abcb1 humanized model express not only the human Abcb1 gene but also has similar expression pattern as human. Conclusions The Abcb1 knock out rat and the Abcb1 humanized rat were successfully established, and this model is close to human concerning about the drug metabolism related to Abcb1.

Abstract:Objective To investigate the effect of chronic restraint stress on sociability in mice. Method Chronic restraint stress was applied to establish stress depression model. The mice were individually housing and restrained for 4 h per day in a 50 mL polypropylene conical tube with ventilation holes. This daily restraint was repeated for 35 consecutive days. The sucrose preference test was applied to evaluate the model. The social approach-avoidance test was used to assess the effect of chronic restraint stress on sociability. Results The sucrose intake was significantly reduced in chronic restraint stress model compared with the control mice (P < 0.01). The control group investigated the social stimulus (stranger cage)significantly longer than the object stimulus(empty cage), thereby demonstrating normal social preference. The model group showed a decrease (sniffing time and frequency)in social investigation (P < 0.05,P < 0.01, vs the control group). The chronic restraint stress group did not demonstrate social preference. The preference index in chronic restraint stress model (ratio time sniffing stranger vs. empty cage) was significantly lower compared with the control mice (P < 0.01). Conclusion The chronic restraint stress showed social avoidance in sociability test.

Abstract:Objective To provide reference of the general physiological index, blood physiology and biochemistry for middle and old-aged cynomolgus monkey. Methods 119 cynomolgus monkey aged from 11～16 (80 were males and 39 were females) were involved in the study. We detected the general physiological index(body weight, the crown-rump length and waist circumference), hematology and blood biochemistry parameters respectively and compared these parameters between males and females. Results Between males and females, there were significant differences in Body weight, the crown-rump length and Waist circumference of the general physiological index(P < 0.01),NEUT#、LYMPH#、EO#、BASO#、NEUT%、LYMPH%、EO%、BASO%、MCV、MCH、RDW-CV、PCT、MONO%、HGB、HCT、MCHC、RDW-SD、MPV of hematology(P < 0.01 or P < 0.05)and TBIL、ALB、GLO、A/G、ALP、GLU、UREA、CREA、TCH、TG、CK、ALT、GGT of blood biochemistry(P < 0.01 or P < 0.05). There were no significant differences in other parameters(P > 0.05). Conclusion It provides reference of general physiological index, blood physiology and biochemistry parameters of cynomolgus monkey and basis for their application in biomedical domain.

Abstract:Objective To amplify the encoding full-length sequence of Tibet mini-pigs myostatin (MSTN) gene and analysis the sequence by bioinformatics software. Method The RNA of liver tissues from Tibet mini-pig was extracted, and reversely transcribed into cDNA. The gene coding region sequence of myostatin gene was amplified through RT-PCR, and then the purified product of PCR was ligated with a pMD19-T and transferred into the bacterium DH5α for replication. The positive clones were screened and sequenced. The sequence characters were analyzed by using bioinformatics method and phylogeny evolution tree was constructed with other twelve species. Results The coding redion of MSTN gene was 1128bp, and coded 375 amino acids. The amino acid homology analysis showed that the homology rate of amino acid sequence was 99%. Conclusions Molecular phylogeny evolution indicated that it had a close relation with human, dog, banna minipig, sheep, goat, cattle, horse, chimpanzee, rat, mouse except chick and zebrafish, and the most closely related with banna minipig.

Abstract:Objective To measure the body weight and body size of the F1 generation in Rongshui miniature pig (RMP). Methods 83 F1 generations of RMPs (48 females and 35 males) were selected randomly. 9 traits included body-weight, body-length, body-height, chest-circumference, chest-breadth, chest-depth, circum of pastern, girth of leg and rictus were measured, and analyzed statistically by SPSS statistical software and Logistic nonlinear growth analysis model. Results In the F1 generations of RMP, the weights of birth day、6^thmonth and 12^th month of female and male were 0.61±0.14 kg and 0.55±0.13 kg, 17.21±5.20 kg and 16.35±5.23 kg, 26.97±6.49 kg and 26.53±5.65 kg respectively. There was no difference significantly between the genders of the 9 measured traits except for born-weight, born-length and chest-breadth in 10^th month (P < 0.05). According to the analysis in Logistic model, body-weight inflection point was between 5^th-6^thmonth, body length and girth of leg inflection points were 2^nd～3^thmonth, while inflection points of other traits were 1^st～2^nd month. Conclusion The F1 generations of RMP showed low mature body weight and sweet temper. It should be breed as good laboratory miniature pig.

Abstract:Objective Assembly whole mitochondrial genome sequence of Rongshui miniature pig (RMP) breed and analysis the structure of mitochondrion based on the next-generation sequecing method. Comparison of phylogenetic relationship and genetic diversity among different pig breeds. Methods We collected peripheral venous blood sample from RMP and constructed two paired-end sequencing libraries. A whole-genome shotgun sequencing strategy and Illumina Genome Analyser sequencing technology were used in our study. Results The mitochondrial genome of RMP consists of 13 protein coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and the length of pig is 16888 bp. The GC content of this pig mitochondrial genome is about 44 %. Based on phlogenetic analysis, population genetic analysis, our findings confirmed that the ancestral cluster in East Asia mainly occurred among Diannan 7# pig, Hainan wild boar, Lanyu and RMP. Conclusion RMP, a typical miniature pig breed in China, is an earlier ancestor than Lanyu pig breed.

Abstract:Objective To detect the blood parameter, blood biochemical and electrolyte indices of the F1 generation of Rongshui miniature pig (RMP). Methods The blood of 43 female and 42 male RMPs of 4^th month old, and 36 RMPs of 12^th month old (half male and female) were extracted from jugular vein. And the blood parameter, blood biochemical and electrolyte indices were detected by blood analyzer and automatic biochemical analyzer. Results In the same month-old RMP, no significant difference between male and female were found in most indices of blood parameter, blood biochemical and electrolyte indices. On the other hand, many indices were difference between 4^th month old and 12^thmonth old RMPs of same gender. Compared with the 4^th month old RMP, the 12^th month old RMP decreased significantly in WBC and PLT, increased in HGB (P < 0.05) while RBC was the same (P > 0.05). Serum ALT, AST, ALP, CK (male), LDH(male), A/G, BUN, GLU (female), CHOL (male) and K⁺ decreased significantly (P < 0.05), while serum TP, TBIL, CR and Ca²⁺ increased significantly (P < 0.05),but serum CHOL, TG, HDL-C and LDL-C were not different. 86.4%(19/22) biochemical and electrolyte indices in RMP were in/or close to the range of normal value of human. Conclusion Most of the blood parameter, blood biochemical and electrolyte indices of RMP were close to human's normal value.

Abstract:Objective To establish the data including anatomy and histology of main organs in Rongshui miniature pig (RMP). Methods F1 Rongshui miniature pigs with male and female (2 in each group) in 6 month old were used in this experiment. We measured body weights, dissected these pigs after anaesthesia, recorded total blood volume, total plasma volume, number of spine and dental formula, took main organs for photographs, and made histological sections observed and took photographs by microscope. Results We gained the photographs of main organs and histological sections, organ weights,organic coefficients and other basic data. Conclusion Basic anatomy and histology data of main organs in RMP were collected.

Abstract:Objective To reduce experimental costs and improve the utilization of S9,we use spiral coating technique to evaluate the activity of rat liver S9 prepared by combination inducing method as well as to establish cryopreservation method. Methods Using spiral coating technique and Ames test to evaluate the activity of self-made rat liver S9 and commercially available S9 separately. We use glycerinum as protective agent to establish cryogenic storage method, so that S9 can be in liquid form stored at -20 ℃ in the refrigerator. Results In the Ames assay as well as using spiral coating technique, the number of revertant colonies had dose-response relationship among the dose of S9. When conditions were the same, the number of revertant colonies in positive control was at the approximate level in presense of self-made rat liver S9 and commercially available S9 respectively. When S9 (concentration of 38%) was added to the amount at 1.48～6.62 μL/μL broth dose of bacteria, it can significantly induced Salmonella typhimurium histidine strains TA100, reverse mutation rates were three times more than the control group. Conclusions Spiral coating technique can successfully evaluate the activity of rat liver S9. The inducing method of combination of PB and BF can take the place of the unducing method of PCB_s in the preparation of Liver homogenate S9.

Abstract:Objective To discriminate whether chemical compounds are micronuclei-inducing by counting the ratio of bone marrow micronucleus polychromatic erythrocytes (MNPCE) dyed with a single fluorescence reagent (acriding organe, AO) by single-laser flow cytometry. Methods Treating male KM mice and SD rat with cyclophosphamide (CP) respectively, and counting their frequencies of micronucleated polychromatic erythrocytes (MNPCE) as well as frequencies of micronucleated normochromatic erythrocytes (MNNCE) in bone marrow by AO and a single_laser flow cytometer (FCM), comparing and analyzing the results from different methods. Results The results showed that, along with increasing dose of CP, the ratio of MNPCE also corresponding increase, suggesting significant quantative-efficiency correlation. MNPCE were also counted manually by fluorescence microscopy, and the results showed no significant difference with that by flow cytometry. Conclusions AO_FCM fully automated detection method for the detection of MNPCE and MNNCE in mice and rat bone marrow micronucleus rate is reliable.

Abstract:Objective To evaluate the efficacy, safety and compatibility of a new type of atrial septal defect (ASD) occluder in atrial spetal defect mini-pigs model. Methods Five Tibet mini-pigs were selected as the ASD models which were established by the combination of atrial septal puncture and balloon dilation. Then the new type occluder was implanted for the therapy of ASD. Transthoracic echocardiography with color Doppler was used in all animals during closure and in follow up examinations. The animals were killed at 3 months after occlusion for electron microscopical observation and microscopic examination. Result The ASD models had been created in five piglets successfully without complication, all of whom were implanted successfully with the new device without shunts. Postmortem and microscopic examination of the 5 specimens 3 months after placement showed complete. Conclusion Transcatheter ASD occlusion with new type ASD occluder is safe,feasible and effective. This occlusion can repair the atrial septal defect successfully.

Abstract:Objective To transfect EGFP gene to porcine embryonic fibroblasts (PEFs) of Tibetan miniature pigs by Lonza Nucleofector II machine and compare the tansfection efficiency between this method and the lipofection method. Method A plasmid carrying green fluorescent protein (GFP) was transfected into PEFs of Tibetan miniature pigs via the Lonza Nucleofector II machine (program U020) and by Lipofectamine 2000. Results 5 hours after nucleofection, green fluorescence was observed, indicating 80% transfecting efficiency in the nucleofection group, which is significantly higher than the lipofection group. Conclusion Nucleofector II machine can efficiently transfect PEFs, provides a reliable method for efficiently generate transgenic Tibetan minipigs.

Abstract:Caenorhabditis elegans (C. elegans), has developed into an useful model for biomedical research, particularly in the targets of novel drugs and the related mechanism. C. elegans, combining with cost-effective cultivation, simple biological structure, make for high-throughput screenings in vivo model. In this review, we explore the feasibility of C. elegans in drug activity evaluation of complex systems. Because of the body independence of C.elegans, it provided good platform for the efficacy and mechanism of research on new drugs as well as Chinese herbal extract in vivo, showing a good prospect.

Abstract:Rat brain development is a complicated process. There are significant changes of histology, cytology and molecular biology in the process of fetal and postnatal brain development. The basic structure of brain has formed in the prenatal period. While the formation of connections between different parts and the improvement of the function of the brain occur after birth, during which many of the variations are bases of the nervous system diseases, so the postnatal brain development is still very important. Although the concept of brain microenvironment has been proposed more than 150 years ago, the research about the changing extracellular space (ECS) during the postnatal brain development has yet to gain significant progress. The author reviewed the anatomical and physiological characteristics of ECS in the process of postnatal development of the rat, stating the important role of ECS in the individual development, which is expected to provide reliable evidences for the explore of mechanisms and effective treatment approaches of pediatric development related nervous system diseases.

Abstract:In recent years many reports on the progress of mammary tumors treated by traditional Chinese medicine (TCM) have appeared in the literature. In this article, progress of clinical and experimental study between human and canine mammary tumors was compared. Ways and methods of how TCM treat mammary tumors were exhibited such as Chinese medicinal formulae, herbal extracts and active ingredients. Meanwhile, mechanisms of TCM treating mammary tumors were pointed out. The purpose of this article is to provide idea about TCM clinical therapy methods for canine mammary tumors, and to provide research foundation and important models for study of human mammary tumors.

Abstract:The provenances of Rongshui miniature pigs (RMPs) were purchased from Rongshui, Guangxi, in 2012. 130 RMPs were transported to Sanshui, Guangdong,China, which were breed according to the laboratory animal standards. 83 RMPs were selected randomly from the first filial generations (F1). The basic data were collected including breed characteristics, reproductive performance, grow curve, hematology, biochemical markers of serum and urine, organ coefficient, Chromosome analysis. According to the national and local standards, the quality control standards of RMP were set up including microbiological, parasitic, environment and facilities, fodder, pathology, genetic. The results showed that RMPs adapt to the climate of Guangdong. The natural mating and conceive rate was 88.3% with the pregnancy of 112 days. The average number of firstborn and still-born was 6.1 and 7.9 respectively. RMP was small body size with the adult body weight of 17.21±5.20 kg and 16.35±5.23 kg in female and male respectively. RMP was very tame. The mitochondrial genome analysis suggested RMP belonged a typical miniature pig breed in China, which is ancient than Lanyu pig. RMP could be breed as a new kind of laboratory animal.