Method for determining the repair activity of non-homologous end joining
The invention discloses a method for determining the repair activity of NHEJ. In this method, HPRT gene is mutated by using a site-directed gene mutation technology, and plasmid transfection, and 6-TG treatment are performed. By means of the method of the invention, the NHEJ repair activity level of cells can be observed by measuring the cell viability. The method can be used for screening the effects of different drugs and different genes on the NHEJ repair activity.
1. A method for determining the repair activity of non-homologous end joining, comprising steps of:
(1) constructing a plasmid for HPRT gene by using a TALEN technology or CRISPR/Cas9 technology;
(2) treating a mammalian cell combined with the constructed plasmid of the step (1) by an NHEJ inhibitor;
(3) culturing the mammalian cell of the step (2) in a culture medium containing 6-thioguanine;
(4) adding a MTT solution into the treated mammalian cell of the step (3) to form blue formazan particles, dissolving the formed blue formazan particles and then determining the light absorption value thereof at OD570 nm via a microplate reader; and
(5) determining the repair activity of non-homologous end joining based on the light absorption value.
2. The method as claimed in claim 1 , wherein in the step (1) when the plasmid for the HPRT gene is constructed using the TALEN technology, the plasmid for the HPRT gene is constructed using a left-arm recognition sequence of a target gene shown in SEQ ID NO:1 and a right-arm recognition sequence of the target gene shown in SEQ ID NO:2.
3. The method as claimed in claim 1 , wherein in the step (1) when the plasmid for the HPRT gene is constructed using the CRISPR/Cas9 technology, the plasmid for the HPRT gene is constructed using a forward primer sequence shown in SEQ ID NO:3 and a reverse primer sequence shown in SEQ ID NO:4.
4. The method as claimed in claim 1 , wherein the mammalian cell of the step (2) is a 293 cell, the NHEJ inhibitor is NU7441 or DNA-PKcs siRNA, and the sequence of the DNA-PKcs siRNA is shown in SEQ ID NO: 5.
5. The method as claimed in claim 4 , wherein the step (2) comprises transfecting the constructed plasmid of the step (1) into the 293T cell, and treating the transfected 293 T cell with the NHEJ inhibitor NU7441.
6. The method as claimed in claim 5 , wherein the plasmid transfection in the step (2) comprises inoculating the 293T cell in a culture dish, and transfecting the constructed plasmid of the step (1) into the 293T cell by using Lipofectamine 3000 as a transfection reagent when the cell density reaches 70%.
7. The method as claimed in claim 4 , wherein the step (2) comprises co-transfecting the constructed plasmid of the step (1) and the DNA-PKcs siRNA into the 293T cell.
8. The method as claimed in claim 7 , wherein the co-transfection of the step (2) comprises inoculating the 293T cell in a culture dish, and co-transfecting the constructed plasmid of the step (1) and DNA-PKcs siRNA into the 293T cell by using Lipofectamine 3000 as a transfection reagent when the cell density reaches 70%.