Abstract:Complex traits are multifactorial traits controlled by polygenic host factors. These trait-related phenotypic characteristics and performance including body weight, blood chemistry, immune cell profiles, as well host susceptibility to infectious and chronic diseases. In recent years, tremendous efforts were invested aiming to map the host genetic factors attribute to these traits and subsequently clone the gene/s underlying these loci. In parallel to human studies, a number of mouse models and approaches were developed aimed to enhance the mapping process and the gene cloning. These include of using resources such as F2, backcross, advanced intercross lines, outbred populations, consomic, congenic and recombinant inbred lines (RIL). The constraints of these approaches were the limited resolution mapping of genomic regions of the quantitative trait loci (QTL) associated with the trait of interests, and the limited genetic diversity observed in the parental founders. To overcome these limitations, a new genetically highly diverse recombinant inbred lines of mouse population was established, namely the Collaborative Cross (CC), created from full reciprocal mating of 8 divergent strains of mice:A/J, C57BL/6J, 129S1/SvImJ, NOD/LtJ, NZO/HiLtJ, CAST/Ei, PWK/PhJ, and WSB/EiJ. By intercrossing these eight founders to generate the different CC lines, the genetic makeup of the newly developed resource is completely different from the eight parental lines, and will show heterosis, which subsequently will response differently comparing with their original founders. Finally, our results suggest that it is not essential to defining the phenotypic response of the eight parental lines, prior of assessing the CC lines, because it is believed that genetic interaction of the new genetic makeup of the new lines will reveal new phenotypic response, which completely different from the parental lines.In this report, we present to the community the power of the CC for dissecting variety of complex traits including host susceptibility to infectious and chronic diseases as well body performance traits. Based on our results from a variety of studies, we recommend to the community, that the best strategy of using this population is to aim of phenotyping about 50 and more of CC lines, with limited number of biological replicates (3-4 mice per line), and subsequently using the publicly available high dense genotype information of the CC lines as well the sequence database of the eight founders, it will be possible performing QTL mapping to a unprecedented precision genomic regions less than 1 MB, subsequently lead to identify potential strong candidate genes. These achievements are believed cannot be obtained with any other currently available mouse resource populations.

Abstract:Animal model plays an important role in prevention and control of infectious disease, which could link basic research in laboratory with clinical diagnosis and treatment for human patients. Mouse is the most widely used animal model of infectious disease, however, adult immunocompetent mice are resistant to some pathogens. The highly genetically diverse Collaborative Cross (CC) mice could recapitulate many of the genetic characteristics of an outbred population, such as humans. Based on this, this review will focus on the application and research progress of CC mice in infectious disease (including viruses, bacteria, fungi etc.), which could provide useful reference data for expansion of animal model resource bank, and implement of precision medicine of major and new emerging infectious diseases. We hope this review could serve as a modest spur to induce other researchers to come forward with their valuable contributions.

Abstract:Cancer is a general term of a series of complex traits of the disease triggered by the body cells losing their normal regulation of excessive proliferation, which essentially is a genetic disease. Recombinant inbred strain (RI) mouse generated from one pair of founders has been widely used in traditional tumor model. However, RI has many limitations on the statistic efficiency because of the small scale and lacking of allele diversity. The Collaborative Cross (CC) was designed to generate hundreds of recombinant inbred lines by 8 divergent strains of mice. CC mice embody a tremendous amount of natural genetic variation in different sub-strains of mouse and the single nucleotide polymorphism is four times of the traditional experimental mice. The high-genetic diversity and large scale population enables CC mice simulates the differences of individual susceptibility to the pathogeny or the therapies,thus provides a better research tool and information platform for expediting discovery of genes and genes function in human complex traits diseases. This review summarizes our current knowledge of this field, including methodologic aspects, applications, challenges and limitations, and utilization for cancer research.

Abstract:The individual variability should be considered in precision medicine-prevention and treatment strategies. Medical research using genomics, proteomics, metabolomics, systems analyses, and other modern tools has made big progress.In 2002, the members of the Complex-Trait Consortium proposed to develop a new mouse genetics resource called the Collaborative Cross (CC). The CC is a genetic reference panel of recombinant inbred lines of mice, designed for the dissection of complex traits and gene networks.It will provide a powerful measure for functional studies of biological networks, which will be essential to understand the intricacies of disease processes.

Abstract:Objective To establish a Chinese hamster model of babesia infection, to find the changing pattern of organs and biochemical parameters in Chinese hamster infected with Babesia, and to promote the detection and treatment of babesiosis.Methods Healthy 5-week old Chinese hamsters were infected by intraperitoneal injection of blood containing Babesia. Blood samples were collected at 0, 2, 4, 6, 8, 10, 12, 14, 16, 23, 30, and 37 days after infection from 5 hamsters at each time point. Blood smears were prepared to detect the parasites using Giemsa staining. ELISA assay was employed to test the IL-2 concentration. The blood biochemical indexes were detected using an automatic biochemical analyzer. DNA was extracted from the whole blood and REAL-TIME RCR was performed to determine the reproduction of Babesia. Aftert the animals were sacrificed, the heart, lung, spleen, liver, and kidney were taken to analyze the changes of organ coefficients. Results The highest level of Babesia in the hamsters occurred on day 4 after the Babesia injection, and then showing a decreasing tendency. However, there was a transient increase on the 12^th day after infection. The liver and spleen displayed most extensive response to the infection showing hepatomegaly and splenomegaly, but the variation of heart and kidneys coefficients was within the norm. There were prominent changes of blood cells, especially leucocytes, with two peaks at day 10 and 23 after the Babesia infection. The peak changes of blood biochemical indexes occurred at day 12 after infection. The concentration of serum IL-2 reached a peak on the 10th day after infection. Conclusions The Chinese hamsters display typical characteristics of tick-borne diseases such as hepatomegaly and splenomegaly. The immunological system is activated along with the infection and reaches a highest stage in the second week. Afterwards the Babesia can live in the hamster body for a long period of time. The results of this study provide useful information supporting further studies on the detection, treatment and prevention of Babesiosis.

Abstract:Objective To investigate the efficiency of target gene transfection of the heart and liver after tail vein or intramyocardial injection of adenovirus vector (GFP-Ad). Methods GFP-AD was constructed at first. A total of 20 male 8-week old C57BL/6 mice were randomly and equally divided into tail vein injection of GFP-AD group and intramyocardial injection of GFP-AD group. The mRNA levels of GFP in the heart and liver tissues were detected by Q-PCR at different time points. Fluorescence microscopy was performed to visualize the expression of GFP fluorescence. Results Compared with the tail vein injection group, the GFP mRNA level in mouse heart tissue was apparently higher in the intramyocardial injection group. In both groups, the GFP mRNA levels in liver tissue were significantly increased compared with that in the heart tissue. In the tail vein injection group, the GFP mRNA level in liver tissue reached a peak on day 7; but in the intramyocardial injection group, the mRNA level of GFP in liver tissue reached apeak on day 3. We also observed the same trend of GFP fluorescence expression in the tail vein injection group compared with that in the intramyocardial injection group.Conclusions Intramyocardial injection of adenovirus vector is suitable to achieve a higher transfection efficiency in mouse heart tissue compared with the tail vein injection method. Although both injection methods are suitable for transfection of mouse liver, the tail vein injection method is preferential for it is simple and less invasive.

Abstract:Objective To prove the advantages of telemetry by comparing with the traditional methods in safety pharmacology. Methods To monitor continuously the heart rate, respiratory rate, blood pressure and ECG of Beagle dogs by traditional and telemetry methods respectively, analyze and compare the changes between anesthetized and conscious dogs before and after feeding. Results Maintenance of anesthesia changed significantly the heart rate, respiratory rate, blood pressure and QT interval in the ECG of animals. The changes of physiological indicators in 24 h is not obvious in conscious animals, and showed a certain biorhythm. Compared with the conscious animals, the anesthetized dogs' heart rate was significantly higher, blood pressure increased significantly, QRS and QTcf interval prolonged significantly, respiratory frequency decreased, heart rate increased significantly after feeding, and QTcf interval extended very significantly. Conclusions Traditional methods in safety pharmacology affect animal physiological indicators such as heart rate, blood pressure, respiratory rate and QT interval, which affect the objectivity of drug evaluation. Using conscious animals by telemetry can reduce these errors, however, the interference from outside should be eliminated.

Abstract:Objective To investigate the dynamic changes of metallothionein(MTs)gene expression and explore the important significance of MTs during hepatocarcinogenesis. Methods One hundred and twenty-five SPF 5-8-week old male C57BL/6J mice were randomly divided into control group and model group. Diethylnitrosamine (DEN) was given to the mice at a dose of 100 mg/kg, ip, and 50 mg/kg, ip, in the first and next week, respectively. The mice were given ethanol (53%, 5 mL/kg/day, 5 days/week) from the third week of experiment till 35 weeks. At 1, 3, 9, 13, 24 and 35 weeks of the experiment, liver samples were taken for histopathological examination of liver damages and incidence of HCC. The liver index and malondialdehyde (MDA) of liver homogenate were determined. All liver tissue samples were examined by histopathology using hematoxylin and eosin (HE), Masson and reticular fiber staining. Real-time RT-PCR was used to analyze the mRNA expression level of liver metallothionein-1/2 (MT-1/2) in different periods. Results Progressive liver damages in model group mice were identified in different periods. Hepatocytes abnormal tission and abnormal liver plate structure, architecture often characteristic of HCC could be seen in approximately 50% of mice at 35 weeks. In addition to these, a higher liver index also were seen at 35 weeks. Increased MDA levels in the mouse liver tissues were observed in each stage. Real-time RT-PCR analysis showed that significantly increased transcription of MT-1 and MT-2 at 1, 3 and 9 weeks, then gradually declined and even below the normal level. Conclusions MTs gene expression levels in mouse liver tissues are changed from significantly increased in the early stage of injury to decreased expression combined with distinct fibrosis. Our findings further demonstrate that the down-regulation of MTs level is closely correlated with hepatocarcinogenesis.

Abstract:Objective To investigate the long-term toxicity of an Uyhgur medicine, Kursi Kaknaq, on hematological parameters in the rats. Methods A total of 120 healthy Sprague-Dawley rats were randomly divided into the control, low dose (0.32 g/kg·d), moderate dose (1.6 g/kg·d) and high dose (3.2g/kg·d) Kursi Kaknaq groups.The drug was given orally, 6 days per week for 180 days. The control group was given 0.5% sodium carboxymethyl cellulose suspension. Results No death was recorded in the rats and no obvious toxic events were observed during the experiment. Hematological parameters including RBC,HGB, MCH, MCHC, LYMP%, WBC, and PLT; biochemical parameters including ALT,AST, ALP, GLU, BUN, ALB,TBIL, Crea, TCHO, TG, and CK; electrolyte time parameters such as K⁺, Na⁺, Cl- and prothrombin time(PT) showed statistically significant differences (P<0.05 and P<0.01), but did not show time and dose effect regularity, and no pathological significance. Conclusions No obvious toxic effects on hematological parameters are observed in the SD rats treated with Kursi Kaknaq at doses of 0.32 g, 1.6 g, or 3.2 g (crude drug)/kg·d orally administered for 180 days, indicating that this drug is safe for long-term clinical use.

Abstract:Objective To investigate the effect of A53T α-synuclein on the expression of type 2 vesicular monoamine transporter (VMAT2) in neuronblastoma SH-SY5Y cells stably expressing A53T α-synuclein. Methods A53T α-synuclein eukaryotic plasmid was constructed by transfection of the SH-SY5Y cells using Lipofectamine^TM2000, and a stable transfected monoclonal cell line was selected by G418. Western blotting and DCFH-DA staining were used to detect the effect of A53T α-synuclein overexpression on the expression of VMAT2 protein and level of reactive oxygen species (ROS). Results Western blotting showed that compared with the control group, the expression of VMAT2 protein was significantly decreased, and DCFH-DA staining showed that DCF signal was significantly increased (507.3±7.1) than that in the cell line stably expressing A53T α-synuclein (410.7±10.5) (P<0.05). Conclusions A53T α-synuclein can increase the intracellular ROS level by inhibiting the expression of VMAT2, thereby playing an important role in the pathogenesis of Parkinson's disease.

Abstract:Objective To determine if aquaporin1 (AQP1) and aquaporin5 (AQP5) are expressed in the alveolar-capillary membrane in rats, and to investigate the changes of AQP1 and AQP5 expression in the rat with acute lung injury. Methods The distribution of AQP1 and AQP5 in alveolar capillary membrane was investigated by immunohistochemistry and immunoelectron microscopy with affinity-purified antibodies to human AQP1 and AQP5. The possibility that alveolar capillary membrane AQP1 and AQP5 undergo altered regulation was studied by a rat model established using intra-tracheal instillation of lipopolysaccharide (LPS). Results Immunolabelling showed that AQP1 was stained primarily in the microvascular endothelium of normal lungs, while AQP5 was expressed in type I pneumocytes. Immunohistochemical analysis showed a significant decrease in the expression of AQP1 and AQP5 in injured lungs at 4-48 h after LPS instillation. AQP1 protein was resumed partly at 24 h after LPS instillation and steroid administration, whereas AQP5 was unchanged. Conclusions The decreased expressions of AQP1 and AQP5 in injured lungs suggest that both of them may play a role in abnormal fluid transportation.

Abstract:Objective To establish a Hr mutant knockout mouse model to study the function of Hr gene. Methods Transcription activator-like effector nucleases (TALENs) technique was used to disrupt the mouse Hr locus,creating heritable mutations that eliminate Hr function to explore the effects of Hr on hair development and provide a good model to study the function of Hr gene.The phenotype of Hr^-/- mice was observed after birth and skin histology of the transgenic mice was studied by light microscopy. Results It was shown that a F0 mouse with the 2-bp deletion in Hr gene ranging from 86 to 87 base pairs was obtained. The male mice with clear deletion of the Hr fragment and with obvious frame shifting were mated with wild-type female mice, and F₁ mice were achieved.The heterozygous males mated with females to generate the F₂ homozygous mice. The first hair coat of Hr^-/- mice developed normally. Beginning from 14 days after birth, however, there was a rapid hair loss. The mices were completely hairless except for a few vibrissae at 30 days. Histologically, two characteristic structures appeared, the utriculus and dermal cyst. Conclusions The results suggest that Hr^-/- mice are successfully created using TALENs, and Hr is important for regulating hair development, which could explain at least in part the hair loss and be applied to study the mechanism of hair growth and development disorder.

Abstract:Objective To investigate the effect of protein phosphatase 5 (PP5) on lipid metabolism in the PP5 knockout (KO) mice. Methods Male PP5 KO and wild type (WT) mice at the age of 6 weeks were used in this study. In order to study the effect of high fat diet (HFD) feeding, the body weight was measured. The liver histology was examined by HE and oil red O staining. To further verify PP5 functions in the adipogenesis, in vitro experiment was carried out using mouse embryonic fibroblasts (MEF). Western blotting and real-time PCR were performed to quantified the expression of lipid metabolism-related genes in the liver tissues.Results Compared with the WT mice, the body weight gain was slower in the KO mice. The size of the lipid droplets was smaller and the quantity was less in the KO mouse liver tissue. In vitro study revealed that the KO mouse MEF cells showed less differentiated adipocytes with smaller lipid droplets than the WT MEF cells. This observation was further confirmed by detecting the expression of adipogenesis-related genes in the HFD liver. The markers of adipocyte differentiation, such as CD36, AP2, PPARγ2, and Glut4, were significantly decreased, while energy expenditure-related markers, such as phosphorylation of GR and expression of UCP1, were significantly increased. Conclusions Protein phosphatase 5 may play a regulatory role in the mouse lipid metabolism through regulating the de-phosphorylation of p-GR and enhancing the expression of UCP1.

Abstract:Objective Lamins are the major components of nuclear lamina underneath the inner nuclear membrane (INM). Lamins express in most cells and are involved in the whole process of growth, also play a major role in cell stability and embryonic development. Mutant in human LMNA gene may lead to a series of disorders, which are similar to progeria or other aging-associate syndrome. In this study, we report a new lmna knockdown animal model generated in our laboratory in order to provide a useful tool for studying laminopathies. Methods Two plasmids tagged to zebrafish lmna gene were designed based on morpholino oligonucleotides technology. Co-microinjected the plasmids into zebrafish embryos to knockdown lmna gene. Imagining and western blot detection were used to identify the mutants. Results Two different proteins, Lamin A/C, were expressed in the zebrafish embryos. Two plasmids lmna-MO and lmna-EGFP-pCS²⁺ were generated and co-microinjected into embryos. The results of imagining and western blot showed that the expression of lmna gene was downregulated in the zebrafish embryos. Conclusions Lamin A/C are expressed in zebrafish. lmna gene can be knocked down by the injection of lmna-MO and lmna-EGFP-pCS²⁺. This new animal model may be a powerful tool for study on laminopathies.

Abstract:Objective To test and analyze the genetic background of highly immunodeficient mice from different sources. Methods Four highly immunodeficient mouse strains from different sources of NOD background were collected. 30 microsatellite DNA sites were detected, and the genotype can be displayed by gel electrophoresis and STR scanning. Results 17 microsatellite sites exhibit polymorphism in 20 mice of the four groups. There were 30 homozygous loci in the mice of groups A and B, and heterozygous in the other two groups. The genetic distance is minimum between groups A and B, showing a higher genetic similarity. Conclusions The genetic backgrounds are different in highly immunodeficient mice from different sources.

Abstract:Objective To provide original reference data for oral ecosystem research, Tibet minipigs, beagle dogs, rhesus monkey, New Zealand white rabbits and Wistar rats were selected to study their respective characteristics of oral microbial mmunities and compared with normal data of humans. Methods Total DNA was extracted from the specimens of oral microbial communities of Tibet minipigs, beagle dogs, rhesus monkey, New Zealand white rabbits and Wistar rats, and used to amplify 16S rRNA V4 fragments with labeled universal primers. The diversity and structure of microbial communities from those animals were compared with that of humans using BIPES and QⅡME analysis after Illumina sequencing of 16S rRNA V4 fragments. Results The richness of the oral microbial communities of humans and the five species of laboratory animals was significantly different (P<0.05). Different species of animals have their own unique oral flora, among which the oral flora of the monkey is the most similar to that of humans. Conclusions Among the five species of laboratory animals, the oral microbial communities of rhesus monkeys and humans have highest similarity. Specifically, the Fusobacterium and Porphyromonas levels of rhesus monkeys is most similar to those of humans. Our findings indicate that rhesus monkeys may be suitable animal model for studies of human oral microbial communities. Tibet minipigs may be suitable animal model for Proteobacteria studies, while beagle dogs may be appropriate for modeling of diseases related to Spirochaetes.