Nyd - Build And Phenotypic Analysis And Rpap3 Sp15 Transgenic Mice And Ttc21a Protein In Tpr Domain Structure In The Study

Background&Purpose: Deaminase gene is an old and new Enzyme super-familywhich has discovered a lot of members varied on the function of CytidineDeamination in RNA and DNA. What’s more, lots of members in this family arerelated to virus infection, tumorigenesis, tumor drug resistance and immune status.The new finding, NYD-SP15which has cytidine deaminase domain and it has closeties with other cytidine deaminase members on evolution. In this research weconstruct overexpression SP15transgenic mice and then detect the DNA, RNA andProtein levels of mice. Then studies how overexpression SP15would effect thedevelopment of mice from the aspect of Physiological and Pathological level. Andthen it illustrates preliminarily the function of NYD-SP15in vivo.Method: Through the construction of NYD-SP15overexpression in transgenic mice,these mice systemic widespread expression of NYD-SP15protein. By mating theseobtained NYD-SP15transgenic mice with C57/BL6mice, we can obtain stablyexpressed mice offspring. As well as using quantitative PCR and Western blottechnique to detect the expression efficiency of NYD-SP15positive mice. Observingthe weight of positive transgenic mice and their morphological changes in long termand then take use of Biochemical Analyzer to detect the change of blood systemindex of mice, and use X-Ray photograph by Carestream MS FX PRO system todetect the change of NYD-SP15transgene mice from the aspect of Physiologicallevel. And pick up these transgene mice which are abnormal physiologicalphenotype then anatomy to take organs to do immunohistochemical testing, and thento observe cell histological changes of these organ slices. Results: So far we have got52NYD-SP15positive transgene mice and when wemake further investigation on2pairs of mice(11#/1#;85#/84~#), we detect thattransgenic mice mRNA generally overexpression compared to the wild-type andthese mice have the most high overexpression in brain and lung. Using the specificantibody-Flag to do western blot experiment on NYD-SP15organs, and we can findNYD-SP15protein general overexpression in transgenic mice while less-expressionin both pancreas and brain tissue. Although the majority of the transgenic mice stillshowed a normal ability to survive at72weeks of age, but we found in its abnormalphysiological changes in the4~#transgenic mice at52weeks and then take use ofsmall animals in vivo Carestream MS FX PRO system for the X-ray photography onthem, the abdomen of4~#mouse is more hypertrophy than wild control; collectmice’s blood to do biochemical analyzer, found that many physiologic indexchanged obviously; anatomical observation find that4~#mice has a large area ofabdomen filled by fat, all organs bigger than wild-type mice of the same age, and thejunction of the stomach and spleen found one additional sarcoma-like substance,by anatomy found it’s similar to the composition of the lipoma; stained tissue slicesobserved liver, lung and additional sarcoma(which proved to be fat) which hasvisible pathological changes; the study found that NYD-SP15had expression willlead to the occurrence of lesions of epithelial tissue, including liver and lung.Conclusion: NYD-SP15transgenic mice have different expression efficiency in thewhole body; not all transgenic mice have abnormal phenotypes, only some of thesepositive transgenic mice will have special physiological and pathological changes;from lesions occur in the4~#positive mouse we can find its obvious abnormalities inphysiologic index. Histopathological slices indicate that liver, lung and fat tissuehave severe histological changes that remind us of NYD-SP15may be associatedwith other cytidine deaminase family members is a potential proto-oncogene. Purpose: Our study was aimed to affirm whether the TPR domain inPRAP3/TTC21A proteins can interact with HSP70. To construct theRPAP3/TTC21A (TPR domain)-containing lentivirus, and investigate the influenceof the overexpression of PRAP3/TTC21A on cell cycle.Methods: RPAP3/TTC21A with other homology proteins were compared by usingsequence analysis tools. The Yeast two-hybrid we used To invest the coaction ofRPAP3/TTC21A with HSP70and the reaction site. A lentivirus expression vectorwas constructed, and infected MCF7cells. Flow cytometry was used to toinvestigate the effect on cell cycle.Results: RPAP3/TTC21A exist among many species and were highly conserved.The interaction was confirmed, which happens between the three-TPR domain andhsp70terminal GPTIEEVD by Yeast two-hybrid. The results of flow cytometryanalysis showed that the overexpression of RPAP3/TTC21A inhibit the cell cycleand increased apoptosis.Conclusions: RPAP3/TTC21A can interact with hsp70and it happens between thethree-TPR domain and hsp70terminal GPTIEEVD. Overexpression ofRPAP3/TTC21A influenced the cell cycle and apoptosis.