Construct,Sort,and Identify High Exression BDNF In Mesenchymal Stem Cell Lines

Objective:Construct immortalized umbilical cord MSC cell lines by Crispr/Cas9 technology which with AAVS1 targeting point and telomerase gene.Cells were isolated by flow cytometry which with high BDNF expression for following experiments.Method:?.Contruct TERT/GFP plasmid and CRISPR/Cas9 plasmid with targeting AAVS1 Get HAL and HAR gene sequences from the human genome which extracted blood,hPGK from PLK0.1 plasmid gene sequences,FlagX3?T2A-bGH from px458 plasmid gene sequences,hTERT from pBABE neo-hTERT plasmid by enzyme.And then connect these gene sequences through a specific order for hTERT/GFP plasmid by bridged?linked?trsanfered.II.Select MSC cells with high expression of BDNF and then passage Collect supernatants of 1-9 generations of MSC cells for BDNF ELIAS detection,and keep all cells with high BDNF expression for further subsequent experiments.III:plasmid co-transfection The hTERT/GFP plasmid and CRISPR/Cas9 plasmid will co-transfect into MSC cells.Detect the MSC with positive TERT/Gfp gene expression by GFP,Detct the expression of hTERT in MCS cells before and after transfection by rt-pcr.IV.Select the MSC cell subset with high BDNF expression from immortalizal cell line Use flow cytometry instrument select TERT positive MSCs and not transfective cells into two 96-well plates respectively,each hole for 1 cells,placed in 37 ?,5% CO2 incubator,observe and expand cell's proliferation.Detect the expression of BDNF in the supernatant by ELISA.RESULT:?.hTERT /GFP plasmids were successfully constructed and sequenced by enzymatic digeste,ligatie,recombinate and transformate.II.the seventh generation cells is the highest generation for expression of BDNF by Elisa,(P< 0.001).Transfect the TERT?CRISPR/Cas9 plasmid into the seventh generation MSC cells,formed and quantitatively analyze the MSCs by RT-PCR,transfect successfully and express stably.III.Get single cell from flow cytometry,expand culture,and the transfection plasmid group(msc-gfp group)was found to have a concentration of 941.51+201.90pg/ml,the highest subgroup of BDNF was 1311.29pg/ml,and the MSC group was 959.23+205.75 pg/ml by elisa(P>0.05).It suggested that the level doesn't change which of BDNF protein from the immortalizated cells.CONCLUSION:Crispr/Cas9 technology and telomerase gene targeting AAVS1 can successfully construct immortalized MSC cells which secret and express stablly the BDNF.