Construction of mouse dynactin- 1-shRNA lentiviral vector and its knockdown efficiency in mouse podocytes
10.3724/SP.J.1008.2012.01077
- Author:
Peng FU
1
Author Information
1. Department of Nephrology, Tongji Hospital, Tongji University School of Medicine
- Publication Type:Journal Article
- Keywords:
Dynactin 1;
Lentivirus;
Podocytes;
RNA interference
- From:
Academic Journal of Second Military Medical University
2012;33(10):1077-1081
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the lentiviralvector carrying shRNA of mouse dynactin-1 and to identify its knockdown efficiency of dynactin-1 by infecting mouse podocytes. Methods Three pairs of shRNA targeting mouse dynactin-1 were synthesized and subcloned into Bgl P{cyrillic} -Hind Sh{cyrillic} digested pENTR/pTER entry vectors. Then the entry vectors were transferred into pLenti X2 Puro destination vector by LR Clonase reaction. All the constructs were verified by restriction enzyme digestion and sequencing. The pLentiX2 Puro/dynactin-1-shRNA vectors and the packaging vectors were co-transfected into 293FT cells to produce dynactin-1-shRNA lentiviruses, which were used to transfect podocytes and the cells were screened by puromycin resistance. The expression of dynactin-1 protein in podcytes was analyzed by Western blotting analysis. Results Restriction enzyme digestion and sequencing analysis confirmed that the dynactin-1-shRNA lentivirus vector was successfully constructed. The podocyte cell lines stably expressing dynactin-1-shRNA were obtained by puromycin selection. Western blotting analysis indicated that dynactin-1 protein expression was down-regulated by dynactin-1-shRNA lentiviral vector in podocytes. Conclusion We have successfully constructed lentiviral vector of dynactin-1-shRNA, which can effectively down-regulate dynactin-1 protein expression, providing a basis for further studying the role of dynactin-1 in podocytes.
