The Possible Effect Of STK15 Gene On The Proliferation Of NIH3T3 And Construction Of Animal Model Of Transgenic Mice Expressing STK15

ObjectiveSTK15/BTAK/Aurora-A kinase, a member of a novel serine /threonine kinase family that includes the prototypic yeast IPL1 and Drosophila aurora kinases, involved in regulating centrosomal and chromosomal segregation, is associated with human carcinogenesis. Overexpression of this gene was shown to induce abnormal centrosome duplication/distribution, aneuploidy, and transformation in cells. In order to investigate the possible effect of STK15 gene on the proliferation of fibroblast (NIH3T3), NIH3T3 cells were transient transfection of pcDNA3.1-STK15 plasmid and pcDNA3.1 vector as control. In order to investigate the relation between STK15 and cancer, I construct the animal model of transgenic mice expressing STK15.MethodsFirst of all, STK15 gene of human was amplified by reverse transcript polymerase chain reaction (RT-PCR) technique from human embryo lung cell line and cloned into pTZ57R/T vector for sequence analysis. Recombinant plasmid pTZ57R/T - STK15 was constructed. The sequence analysis showed that the gene was consistent with that of GeneBank in predicted amino acid. The STK15 gene was subcloned into the expression vector pcDNA3.1. pcDNA3.1 and pTZ57R/T - STK15 were simultaneously digested by BamH I and Xba I, then purified, ligased, and transformated to construct the recombinant plasmid pcDNA3.1-STK15. The constructed recombinant plasmid which contains the inserted sequence was identified by the restricted enzymes and the sequence analysis. NIH3T3 cells were transient transfection of pcDNA3.1-STK15 plasmid and pcDNA3.1 vector as control. The transfection effects of STK15 gene on the mRNA levels were examined by reverse transcription PCR (RT - PCR), and the transfection effects of STK15 gene on the protein levels were examined by Western blot and Immunocellar chemistry analysis respectively. Cell proliferation rate and viability were measured by MTT. Reconstituted basement membrane invasion assay was utilized to evaluate the cell invasive. Moreover, pcDNA3.1-STK15 expression plasmid was digested by BgI II and Bst1 107 I, then purified, pcDNA3.1-STK15 injection fragment was obtained. Transgenic mice were established by microinjection genes of the DNA fragments into mice pronuclear and the genome DNA of transgenic mice was identified with PCR and Southern blot. Expression of STK15 in the tissue of transgenic mice was evaluated by RT-PCR.ResultThrough digested and sequenced, the inserted DNA sequence was identified the gene of STK15, which shows the construction of pcDNA3.1-STK15 successfully. pcDNA3.1-STK15 plasmid and pcDNA3.1 vector were transfected into NIH3T3 by lipofectamine. STK15 mRNA and protein expression were obvious upregulated at 48 h in pcDNA3.1-STK15 plasmid transfected NIH3T3 cells than control vector transfected cells, which illuminates cell transfection of pcDNA3.1-STK15 successfully. MTT incorporation rate were increased significantly was apparent in pcDNA3.1 -STK15 plasmid transfected NIH3T3 cells. Transwell migration assay results showed NIH3T3 cells migration of pcDNA3.1 -STK15 plasmid transfection was increased significantly compared with vector pcDNA3.1 transfected NIH3T3 cells. Meanwhile, the achievement rate of microinjection was 77%, 907 ova were transplanted into 30 pseudopregnant mice recipients, 106 offspring new born from the ova microinjected. Among the 106 new born mice, 3 were tested to be STK15 positive with PCR while Southern blotting identified only 1 STK15 positive mouse. 3 positve founder transgenic mice could inherit STK15 gene into their F₁ and F₂ generation proved by PCR, PCR conquence showed 2 F₁ transgenic positive mice and 3 F₂ transgenic positive mice, which were conformed that transgene could be possed to its descendants. STK15 expression of testile was positive by RT-PCRConclusionTransient transfection NIH3T3 of pcDNA3.1-STK15 plasmid can lead to upregulate STK15 mRNA and protein expression obviously, which showed transfection NIH3T3 of pcDNA3.1-STK15 successfully. MTT incorporation rate were increased significantly was apparent in pcDNA3.1 -STK15 plasmid transfected NIH3T3 cells and Transwell migration assay results showed NIH3T3 cells migration of pcDNA3.1 -STK15 plasmid transfection were increased significantly compared with vector pcDNA3.1 transfected NIH3T3 cells, which gives rise to cancer.pcDNA3.1-STK15 fragment can integrate into mice genomic DNA. An animal model of transgenic mice expressing STK15 can be successfully established.