Characterization and phylogenetic analysis of Krüppel-like transcription factor (KLF) gene family in tree shrews (Tupaia belangeri chinensis).

Krüppel-like factors (KLFs) are a family of zinc finger transcription factors regulating embryonic development and diseases. The phylogenetics of KLFs has not been studied in tree shrews, an animal lineage with a closer relationship to primates than rodents. Here, we identified 17 KLFs from Chinese tree shrew (Tupaia belangeri chinensis). KLF proteins are highly conserved among humans, monkeys, rats, mice and tree shrews compared to zebrafish and chickens. The CtBP binding site, Sin3A binding site and nuclear localization signals are largely conserved between tree shrews and human beings. Tupaia belangeri (Tb) KLF5 contains several conserved post-transcriptional modification motifs. Moreover, the mRNA and protein expression patterns of multiple tbKLFs are tissue-specific . TbKLF5, like hKLF5, significantly promotes NIH3T3 cell proliferation in vitro. These results provide insight for future studies regarding the structure and function of the tbKLF gene family.

Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution.

The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (∼ 7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency.

The complete mitochondrial genome sequence of the Daweishan Mini chicken.

Daweishan Mini chicken is a valuable chicken breed in China. In this study, the complete mitochondrial genome sequence of Daweishan Mini chicken using PCR amplification, sequencing and assembling has been obtained for the first time. The total length of the mitochondrial genome was 16,785 bp, with the base composition of 30.26% A, 23.73% T, 32.51% C, 13.51% G. It contained 37 genes (2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes) and a major non-coding control region (D-loop region). The protein start codons are ATG, except for COX1 that begins with GTG. The complete mitochondrial genome sequence of Daweishan Mini chicken provides an important data set for further investigation on the phylogenetic relationships within Gallus gallus.

Construction of Anti-CD20 Single-Chain Antibody-CD28-CD137-TCRζ Recombinant Genetic Modified T Cells and its Treatment Effect on B Cell Lymphoma.

BACKGROUND: Immunotherapy has been explored as a new therapy for B cell lymphoma, which is a non-Hodgkin's lymphoma. Because CD20 is a B lymphocyte-specific marker, anti-CD20 single chain-tagged T lymphocytes have already begun to be experimentally used in B cell lymphoma treatment, but its use is still limited because of its unspecific targeting. T cells transfected with CD28 and CD137 can significantly improve the ability of cytokines secretion and anti-tumor effect, as well as extending T cell survival time and improving their proliferation ability. MATERIAL AND METHODS: Genes containing anti-CD20-CD28-CD137-TCRζ were constructed. After cloning and sequencing, the plasmid was constructed and packaged by lentivirus. It was transfected to the peripheral blood T lymphocyte after identification transfection to induce the fusion protein expression. The cells were incubated with Raji cells and the LDH test was performed to detect the cytotoxic effect of CAR-T cells; the tumor volume and survival rate were measured to observe its inhibitory effect on B cell lymphoma in nude mice. RESULTS: Gene with anti-CD20-CD28-CD137-TCRζ was successfully constructed and transfected to the T cell surface. LDH assay revealed that CAR-T cells can kill the Raji cells with a killing rate of 32.89±6.26%. It can significantly inhibit B cell lymphoma growth in nude mice. CONCLUSIONS: T lymphocytes transfected with anti-CD20-CD28-CD137-TCRζ fusion gene can kill B cell lymphoma, which could provide a new strategy for tumor treatment.

PTEN/PIK3CA genes are frequently mutated in spontaneous and medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumours of tree shrews.

Tree shrew has increasingly become an attractive experimental animal model for human diseases, particularly for breast cancer due to spontaneous breast tumours and their close relationship to primates and by extension to humans. However, neither normal mammary glands nor breast tumours have been well characterised in the Chinese tree shrew (Tupaia belangeri chinensis). In this study, normal mammary glands from four different developmental stages and 18 spontaneous breast tumours were analysed. Haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) showed that normal mammary gland morphology and structures of tree shrews were quite similar to those found in humans. Spontaneous breast tumours of tree shrews were identified as being intraductal papilloma, papillary carcinoma, and invasive ductal carcinoma with or without lung metastasis. To further analyse breast cancer tumours among tree shrews, 40 3-4 month-old female tree shrews were orally administrated 20 mg 7,12-dimethylbenz(a)anthracene (DMBA) or peanut oil thrice, and then, 15 of these DMBA administrated tree shrews were implanted with medroxyprogesterone acetate (MPA) pellets. DMBA was shown to induce breast tumours (12%) while the addition of MPA increased the tumour incidence (50%). Of these, three induced breast tumours were intraductal papillary carcinomas and one was invasive ductal carcinoma (IDC). The PTEN/PIK3CA (phosphatase and tensin homologue/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), but not TP53 and GATA3, genes are frequently mutated in breast tumours, and the PTEN/PIK3CA gene mutation status correlated with the expression of pAKT in tree shrew breast tumours. These results suggest that tree shrews may be a promising animal model for a subset of human breast cancers with PTEN/PIK3CA gene mutations.

[Measurement and analysis of anatomical parameter values in tree shrews].

Anatomical parameter values in tree shrews are major biological characteristic indicators in laboratory animals. Body size, bones and mammilla, organ weights, coefficient intestinal canal and other anatomical data were measured and analyzed in laboratory domesticated tree shrews (7 to 9 months of age). Measurement of 31 anatomical parameters showed that body height, width of the right ear, ileum and colon had significant differences between males and females (P<0.05). Highly significant differences were also found in body slanting length, chest depth, torso length, left and right forelimb length, right hind limb length, left and right ear length, left ear width, keel bone length, left and right tibia length, duodenum and jejunum (P<0.01). With body length as the dependent variable, and tail length, torso length, right and left forelimb length, and left and right hind limb length as independent variables for stepwise regression analysis, the regression equation for body length = 13.90 + tail length × 0.16. The results of 37 organs weights between female and male tree shrews showed very significant differences (P<0.01) for weight of heart, lungs, spleen, left and right kidney, bladder, left and right hippocampus, left submandibular gland, and left and right thyroid gland, as well as significant (P<0.05) differences in the small intestine, right submandibular gland, and left adrenal gland. The coefficient of heart, lung, stomach, bladder, small and large intestine, brain, right hippocampus, and left adrenal gland showed highly significant differences (P<0.01), while differences in the right kidney, left hippocampus, left submandibular gland, right adrenal gland, and left and right thyroid gland were significant (P<0.05). With animal weight as the dependent variable and indicators of heart, lung, liver, spleen, left and right kidney and brain as independent variables for stepwise regression analysis, the regression equation showed that weight = 62.73 + left kidney × 79.21 + heart × 24.09. Female and male laboratory domesticated tree shrews showed certain influences in body size, organ weight and coefficient, and intestinal canal regarding anatomical parameters. This experiment provides basic data for studies on laboratory tree shrews and animal models.

BDNF expression with functional improvement in transected spinal cord treated with neural stem cells in adult rats.

Neural stem cells (NSC) could promote the repair after spinal cord transection (SCT), the underlying mechanism, however, still keeps to be defined. This study reported that NSC grafts significantly improved sensory and locomotor functions in adult rats with SCT in acute stage after injury. NSC could survive; differentiate towards neurons or glia lineage in vitro and vivo. Biotin dextran amine (BDA) tracing showed that little CST regeneration in the injury site, while SEP was recorded in NSC engrafted rats. Immunohistochemistry and Real time PCR confirmed that engrafted NSC expressed BDNF and increased the level of BDNF mRNA in injured site following transplantation. The present data therefore suggested that the functional recovery following SCT with NSC transplantation was correlated with the expression of BDNF, indicating the usage of BDNF with NSC transplantation in the treatment of SCI following injury.

Characterization of spontaneous breast tumor in tree shrews (Tupaia belangeri chinenesis).

Breast cancer is a common malignant tumor. It is essential to develop suitable animal models for discovering novel preventive and therapeutic approaches. Tree shrews (Tupaia belangeri chinensis) have a closer evolutionary relationship with humans than do rodents, which have been widely used in laboratory research. Spontaneous breast tumors were identified in tree shrews in 1960s; however, no detailed studies about tree shrew breast tumors have been conducted to date. Here, we characterized a spontaneous breast tumor from tree shrews by Haematoxylin Eosin (H&E) staining. This tumor was identified as a papillary tumor. Immunohistochemical staining (IHC) for progesterone receptor (PR), Ki-67 and cleaved caspase-3 showed that tumor cells were positive for PR, highly proliferative, and less apoptotic compared to normal breast epithelial cells. Thus, the spontaneous tumor of tree shrew is very close to human papillary tumors in terms of morphology and pathology and we concluded that tree shrew may be a suitable animal model for breast cancer research.

[Progress and prospect in research on laboratory tree shrew in China].

We outline the historical research on the laboratory tree shrew in China and discuss its current research trends. Five key aspects of applied research are emphasized in this review, including quality control standards for laboratory tree shrews, the establishment of an inbred colony, commercial preparation of major molecular and cellular research tools, further research on tree shrew models for human diseases, and the establishment of the tree shrew seed institution at state level.