Construction And Identification Of Lentiviral Mediated RNA Interference Of Mouse Metastasis Suppressor 1 Gene

Metastasis suppressor 1 (Mtss1), namely Missing in metastasis (MIM), has been characterized as an actin-binding scaffold protein that may be involved in cancer metastasis. It has been reported that Mtss 1 mainly participates in the regulation of the microfilament system. Its over-expression triggers distinct cell shape changes such as increase in the formation of lamellipodia-like or filopodia-like structure, accompanied by the reduction of stress fiber. Moreover, over-expression of Mtss1 influenced cell movement and related dorsal ruffling structure. Signaling pathway study showed that it was involved in src kinases and the small GTPase Rac-mediated regulation of cell morphology and cell motility.However, those reports could not answer a question, i.e., what is the function of endogenous Mtss 1? As developmentally regulated protein, endogenous expression of Mtss 1 expression is very low in the adult or fully differentiated cells. As the result, almost all cell lines used in its functional study had very low endogenous expression level and the major research data were achieved from over-expression technique.Fortunately, there is an exception that endogenous Mtssl remains a moderate to high expression level in the purkinje neuron of adult animal, which brought a ray of hope to the functional study of endogenous Mtss1. In the central nervous system, purkinje neuron possesses the most complex dendritic structure. In the process of neural morphogenesis and plasticity, microfilament system plays a major role. Usually, cognitive disorder neurological diseases and neurodegenerative diseases associated with the abnormality of cytoskeleton protein. The reports and our unpublished data showed that Mtss1 have an expression peak around two weeks after birth and maintain a moderate level in adult. Further cellular distribution study reveled that Mtss1 were prominent in the cell body, axon and dendrites, indicating its association with neuronal morphogenesis.In contrast with over-expression technique, application of knock-down and knock-out technologies lead to the reduction or the absence of the targets. Results from above strategies could speak for each other. Compared with the knock-out, knock-down were favored by lots of researchers for its easy-to-use and low cost.RNA interference technique (RNAi), worked through the introduction of double-stranded RNA, could specifically induce the degradation of targeted sequence, finally the gene silencing at post-transcriptional level. As a highly efficiency and specific knock-down technology, RNAi has been widely used in recent study.Lentivirus introduced RNAi could be used in the knock down of the non-mitotic cell like neuron with high efficiency. It could meet the requirements in our study.For the preparation of Mtssl RNAi lentivirus with high efficiency, Invitrogen online design software BLOCK-ITTM RNAi Designer (https://rnaidesigner.invitrogen.com/rnai) have been used to design three pairs of specific shRNA sequences targeting mouse Mtss1 gene. Long oligomers were synthesis by the Sangon Bio-tech (Shanghai) Co., Ltd.To determine the knock-down efficiency of the sequence, three pair of oligomes were ligated into retrovirus vector pSUPER-retro-puro and the constructs were verified by double-digestion and sequencing. After that, the pSUPER-Mtssl-shRNA plamids were transiently transfected into GFP-Mtss1 over-expressed stable cell line followed by western blot. As caculated, the RNAi efficiency of these targeted sequence are more than 60%.After verifying the efficiency of target sequence, DNA fragments including targeted sequences in pSUPER-Mtssl-shRNA were released by EcoRI-Clal double-digestion, cloned into lentiviral vector followed with verification by double-digestion and sequencing.Then, Mtss1 shRNA lentiviral vector, envelope protein plasmid pMd2.G and packaging vector psPAX2 were co-transfected into 293T packaging cell for the harvest of Mtss1 shRNA lentivirus. After purification, the titers of the lentivirus were determined by infect GFP-Mtss1 over-expressed cell lines. The western Blotting results showed that the RNAi efficiency of shRNA lentivirus was over 60%.To test whether the lentivirus prepared above could infect purkinje neuron effectively, primary cultured purkinjie neurons have been used and resultantly immuno-fluorescent assay suggested successful infection. Due to the limited number of purkinjie neurons and relative long culture period, the caculated knock-down efficiency of Mtss1 mediated by lentivirus and related phenotype change remains ongoing.In summary, this work is critical for the in vivo study of Mtss1 in the morphogenesis of purkinje neuron. It has established a fundamental and essential foundation for the study in near future.