Preliminary Study Of SMYD3 Gene Knockout In Bel-7402 Cells Mediated By CRISPR/Cas9

Objective:1.Design three sgRNA targeting SMYD3 gene.They are respectively recorded as sgRNA#1,sgRNA#2 and sgRNA#3.2.Construct the recombinant plasmids of sgRNA#1-PX459,sg RNA#2-PX459 and sgRNA#3-PX459.3.Detecte the expression of SMYD3 in HEK293 T cells and Bel-7402 cells.4.Explore the best package proportion of Lipofectamine 2000 to 1 ?g sgRNA-PX459 of recombinant plasmid.5.Compare the toxity of different concentrations of purinamycin to HEK293 T cells and Bel-7402 cells respectively,and determine the minimum lethal concentration in the third days.6.Compare the activity of the CRISPR/Cas9 targeting carrier.7.Prepare the SMYD3 gene deficient Bel-7402 cell line.8.Compare the proliferation ability of SMYD3 gene deficient Bel-7402 cell line with wild type Bel-7402 cell line.9.Compare the migration ability of SMYD3 gene deficient Bel-7402 cell line with wild type Bel-7402 cell line.Methods:1.Search for the SMYD3 gene sequence on the GenBank site.And design the sgRNA of SMYD3 gene on the http://crispr.mit.edu/ site.2.PX459 plasmids were linearized,sgRNA was connected into the linearized PX459,and the recombinant plasmid of sgRNA-PX459 was constructed.3.With different volumes of Lipofectamine? 2000,1 ?g sg RNA#1-PX459,sgRNA#2-PX45 and sgRNA#3-PX459 recombinant plasmids were packaged,respectively.The best proportion of package is determined by agarose gel electrophoresis.4.With different concentrations of puromycin,HEK293 T cells and Bel-7402 cells were treated for three days,respectively.The minimum lethal concentration was determined after three days by microscopic observation.5.HEK293 T cells were transfected with sgRNA#1-PX459,sgRNA#2-PX459 and sgRNA#3-PX459 recombinant plasmids respectively.The PCR product was sequenced and the activity of CRISPR/Cas9 targeting carrier was determined.6.The Bel-7402 monoclonal cell line was obtained by the limit dilution method.7.The expression of SMYD3 protein in the monoclonal cell line was detected by Western Blot.8.Bel-7402 monoclonal cell lines without SMYD3 protein expression were sequenced by PCR products connected with TA vector and the SMYD3 gene deficient Bel-7402 monoclonal cell line was determined.9.Compare the proliferation ability of SMYD3 gene deficient Bel-7402 cell line with wild type Bel-7402 cell line by CCK-8 test.10.Compare the migration ability of the SMYD3 gene deficient Bel-7402 cell line with the wild type Bel-7402 cell line by the scratch test.Results:1.Three sg RNA targeting SMYD3 gene were designed.The sequences were as follows:2.The best package proportion of sgRNA#1-PX459 to Lipofectamine? 2000 is 1 ?g:3 ?l.The best package proportion of sgRNA#2-PX459 to Lipofectamine? 2000 is 1 ?g:3 ?l.The best package proportion of sgRNA#3-PX459 to Lipofectamine? 2000 is 1 ?g:3 ?l.3.When different concentrations of puromycin was applied to HEK293 T cells and Bel-7402 cells for three days,the lowest lethal concentration of HEK293 T cells was 2 ?g/ml,and the lowest lethal concentration of Bel-7402 cells was 17 ?g/ml.4.The PCR product was sequenced and the activity of CRISPR/Cas9 targeting carrier was determined.The results showed that sgRNA#1-PX459 was active,and sgRNA#2-PX459 and sg RNA#3-PX459 were inactive.5.A total of 42 Bel-7402 monoclonal cell lines were obtained by the limit dilution method.6.Western Blot detection showed that the number #23 cell line had no SMYD3 protein expression.7.Sequencing results showed that there was only one sequencing type of the coded #23 cell line,and the SMYD3 gene defective Bel-7402 monoclonal cell line was obtained.8.Scratch test showed that the migration ability of SMYD3 gene deficient Bel-7402 cells was weaker than that of wild type Bel-7402 cells.9.CCK-8 experiments showed that the proliferation ability of SMYD3 gene deficient Bel-7402 cells was weaker than that of wild type Bel-7402 cells.Conclusion:1.A recombinant plasmid sgRNA#1-PX459 targeting SMYD3 gene targeting vector was successfully constructed.2.The results of TA cloning sequencing and Western Blot showed that the number #23 monoclonal cell line was a SMYD3 gene deficient Bel-7402 cell line.3.Compared with the wild type Bel-7402 cells,the proliferation and the migration ability of the SMYD3 gene deficient Bel-7402 cells weakened.