Abstract:

Objective

To construct the Cdc6-targeted RNAi lentiviral vector plasmids.

Methods

Five targets aimed at Cdc6 （Target 1， 2， 3， 4， 5） were designed by the EASY^TM siRNA software.The Cdc6 siRNA expression plasmids and the Cdc6 eukaryotic over-expression vector plasmids were constructed and used to co-transfect 293T cells.Western blot was used to detect the expression of Cdc6 protein.The interference effects of different targets were evaluated，and the best two sequences were selected to prepare the Cdc6-targeted RNAi lentiviral vectors.Tca8113 cells were transfected with Cdc6-targeted RNAi lentiviral vectors， Real time RT-PCR and Western blot were used to examine the knock-down effects on the levels of expression of Cdc6 mRNA and protein of Tca8113 cells.The MTT method was used to examine the growth levels of Tca8113 cells.

Results

The Cdc6 siRNA expression plasmids and the Cdc6 over-expression vector plasmids were successfully constructed.The exogenous target screening experiments showed that target 3 and target 4 had significant knock-down effects on the expression of Cdc6 protein，compared with the NC group，The knock-down efficiencies were 69.15% and 84.85% ，respectively.The result of RT-PCR and Western blot showed that the Cdc6-targeted lentiviral vector KD1 and KD2 groups had significant knock-down effects on the expression of Cdc6 mRNA and protein of Tca8113 cells.Compared with the NC group， the knock-down efficiencies of Cdc6 mRNA in the KD1 and KD2 groups were 50% and 65%， respectively.The levels of expression of Cdc6 protein were reduced by 65.87% and 79.38% in the KD1 and KD2 groups， respectively.The cell growth levels in the KD group were inhibited， the cell growth inhibition rates in the KD1 and KD2 groups were 25.84% and 30.34%， respectively.

Conclusion

The Cdc6-targeted RNAi lentiviral vectors plasmids， Target 3 and Target 4， were successfully constructed.

Key words: Cell division cycle protein 6, RNA interference, Lentiviral vector