Construction Of A Stable Mouse Fibroblast Cell Line And Screening Of Effective Targets By Down-regulation Of MTOR

PurposeTo establish a mouse fibroblast NIH/3T3cell line which can stably inhibit the expression of mTOR gene, to provide a cell model for studying the function of AKT/mTOR pathway in ARMD and to observe the influence of mTOR knockdown on related proteins.MethodsThree shRNA interference fragments were designed and synthesized based on murine mTOR gene, shl antisense:AGTACTGTAGCACCT; sh2antisense: TCTTCTCTCTGTAGTCCCG; sh3antisense:TGACAAGGAGATAGAACGG. All of these double-strand shRNA hairpins were separately cloned into PIGZ-GFP+Puro vectors to produce lentiviral vector plasmids. The lentiviral vector plasmids and lentiviral packaging plasmids were co-transfected into NIH/3T3cells to package the lentivirus. Evaluate the infection rate with fluorescence microscope. After puromycin selection and culture expansion, stable cell clones were established. After extraction of the RNA by Trizol method and reverse transcription for cDNA, the expression of mTOR gene was tested by real-time PCR. Totally extract the cell protein and test its concentration by BCA method. Evaluate the mTOR gene expression at protien level in NIH/3T3cells by western blot to get the shRNA with the most efficient RNA interference.Results1、The expression of mTOR gene in mouse fibroblast NIH/3T3cell line. The result of PCR showed a highlghted strip at the184bp, illustrating the expression of mTOR gene was enough. The single strip showed perfect specificity without inpuritied. Similar results could be found in western blot test which the mTOR protien showed darkly with single product. Both result from PCR and western blot showed perfect expression of mTOR gene in mouse fibroblast NIH/3T3cell line.2、Results of lentivirus infection and puromycin selection under fluorescence microscope. After3days’infection, infection rate reached more than90%in all groups. The stable cell line was restablished when the cells in the control group died totally by puromycin selection after3to6days.3、Selection results of effective interference fragment. The real-time PCR showed that the statistical correction result for shl, sh2, sh3, negative control and blank control group were respectively0.687±0.131,0.682±0.079,0.547±0.066,0.996±0.036,1.00under lower multiplicity of infection (MOI);0.529±0.073,0.435±0.090,0.284±0.028,0.808±0.120,1.00under higher MOI. Comparing with blank control group, the knockdown rate for shl, sh2and sh3were respectively31.3%,31.8%and45.3%in lower MOI group, while47.1%,56.5%and71.6%in higher MOI group. The mTOR gene was lowerly expressed at protien level in NIH/3T3cell line for sh1, sh2and sh3. ConclusionsIn this current study, three interference targets and one negative control were designed targeting the mouse mRNA of mTOR gene. After exclusion of the interference from the transfected sequence itsself, I successfully established lentiviral vector for mTOR shRNA. With lentiviral plasmids transfection, puromycin selection and culture expansion, I got the cell clones stablely infected with lentiviral vectors. The method I used and established was excellent because shRNA can effectively infect the NIH/3T3cell line and suppress the expression of mTOR which can provide a cell model for studying the function of AKT/mTOR pathway in ARMD.