Preliminary Study Of RNA Interference On The Expression Of MPcdh18Gene In Vitro

Protocadherins constitute the largest subgroup within the cadherin family of calcium-dependent cell-cell adhesion molecules. Roles in tissue morphogenesis and formation of neuronal circuits during early vertebrate development have been inferred. In the postnatal brain, protocadherins are possibly involved in the modulation of synaptic transmission and the generation of specific synaptic connections.Protocadherin-18belongs to the82-protocadherins. Studies had shown that down regulation of Pcdh18induced abnormal embryonic neurogenesis and caused fetal death in a dose-dependent fashion in zebrafish, which provided clues to determine the gene function of human Pcdh18. However, the homology of Pcdhl8nucleotide sequence in zebrafish and human is only66%, and their protein structures are not exactly the same. These differences made it necessary to study Pcdh18in mammalian models to get more reliable information for determining the gene function.As the data suggested, Pcdh18was essential for embryonic survival and loss of Pcdh18resulted in fetal death mainly because of neurogenesis defects. Thus, it was not optimistic to obtain the Pcdhl8knockout mice by traditional knockout technology for further research. RNA interference(RNAi) is a general mechanism for silencing the transcripts of active genes, and this process of post-translational gene silence can be initiated by small interfering RNA(siRNA). Combination of RNA interference (RNAi) theory and transgenic mice technology, a sound method was to construct Pcdhl8RNAi mice in which Pcdh18was properly knockdown but not knockout by controlling the rate of gene inhibition. The advantage of the RNAi mice model is to avoid fetal death, as well as to produce defective phenotype for researching the gene function. This study aims at screening out suitable siRNA sequences which can effectively inhibit the expression of mPcdh18gene through cell culture in vitro, so as to provide appropriate siRNA for the RNAi mice. The experiment performed with the following three aspects.1. Construction and identification of the fusion expression vector pEGFP-mPcdh18Firstly, we obtained mPcdh18gene sequences (lack of termination codon) from the14.5d’s mouse embryos with RT-PCR, and then it was identified by base sequencing. The amplified products were directionally cloned into the prokaryotic expression vector pEGFP-N1, in-frame fusion in the N-terminal domain of the EGFP gene. The constructed recombinant plasmid was selected and identified by double enzyme digesting and sequencing, and the correct fusion expression vector was named as pEGFP-mPcdh18. It was then transfected into mouse fibroblasts NIH/3T3and human embryonic kidney cells293T, and the fusion expression of mPcdh18gene was observed by fluorescence microscope and RT-PCR. Detection of subcellular localization of mPcdhl8through fluorescence microscope shows that Pcdh18is mainly expressed in the cytoplasm with character of dot and is relatively more injunctions of cells. The results provide a reference point for further research of the gene function of mPcdhl8at a cellular level, and the generated recombinant vector can be used for further investigation on RNA interference.2. Construction and expression of the eukaryotic recombinant vector pcDNA3.1(+)-mPcdhl8The amplified products were directionally cloned into the eukaryotic expression vector pcDNA3.1(+), and then the constructed recombinant plasmid was selected and identified by double enzyme digesting and sequencing. The correct recombinant vector was named as pcDNA3.1(+)-mPcdh18. It was then transfected into the Vero cells (the kidney cells of the African green monkey), and the protein expression of mPcdh18gene was detected by cell immunohistochemistry, indirect immunofluorescence assay and RT-PCR. The results showed that the eukaryotic expression vector pcDNA3.1-mPcdh18was successfully constructed, and its expression in COS-7cells demonstrated once again that Pcdh18is mainly expressed in the cytoplasm. Meanwhile the constructed eukaryotic expression system will provide a new experimental material for further studying the protein expression and cell adhesion function of mPcdh18.3. Prediction analysis of siRNA sequence and the screening of high-efficiency siRNA inhibiting the fusion expression of mPcdh18Candidate siRNA targeting mPcdhl8gene (siRNA-mPcdh18) was designed and chemically synthesized. Four pairs of siRNA sequence were designed for specific interfering in mPcdh18. To investigate the inhibition effect of siRNA-mPcdh18, pEGFP-mPcdh18and siRNA were co-transfected into293T cells, and the green fluorescence was observed by fluorescence microscope after transfection. The green fluorescence in293T cells co-transfected with pEGFP-mPcdh18and siRNA were90%fewer than that of negative control by flow cytometry, which show that the siRNA-mPcdh18can effectively silence the extrinsic mPcdhl8gene in293T cells during48-72h after transfection.In summary, this study has successfully constructed the fusion expression vector pEGFP-mPcdh18and the eukaryotic recombinant vector pcDNA3.1(+)-mPcdh18that can highly express in mammalian cells, and screened out high-efficiency siRNA sequence that can inhibit the fusion expression of mPcdh18by cell culture in vitro. This would provide a potential tool for further study in gene function of mPcdh18.