Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (40): 7541-7544.doi: 10.3969/j.issn.1673-8225.2011.40.030

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Construction of Nesprin genome RNAi lentiviral vector

Yang Wen-gang, Xue Song, Lian Feng, Wang Yong-yi, Huang Ri-tai, Zhu Hong-sheng   

  1. Department of Cardiothoracic Surgery, Renji Hospital Affiliated to Medical College of Shanghai Jiao Tong University, Shanghai  200127, Jiangsu Province, China
  • Received:2011-07-05 Revised:2011-08-06 Online:2011-10-01 Published:2011-10-01
  • Contact: Xue Song, Chief physician, Doctoral supervisor, Department of Cardiothoracic Surgery, Renji Hospital Affiliated to Medical College of Shanghai Jiao Tong University, Shanghai 200127, Jiangsu Province, China XUESONG64@163.com
  • About author:Yang Wen-gang★, Master, Physician, Department of Cardiothoracic Surgery, Renji Hospital Affiliated to Medical College of Shanghai Jiao Tong University, Shanghai 200127, Jiangsu Province, China ywgada@hotmail.com
  • Supported by:

    Shanghai Science and Technology Committee, No. 064119636*

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Abstract:

BACKGROUND: Nesprin protein absence can affect the cytoskeletal tissue and homeostasis, causing the loss of cytoskeleton rigidity or leading to premature aging of cells. In this study, we investigated the effect of Nesprin protein on bone marrow mesenchymal stem cells through designing Nesprin siRNA lentiviral vector.
OBJECTIVE: To construct Nesprin-siRNA lentiviral vector.
METHODS: According to the target gene sequence of Nesprin, four pairs of miRNA oligo were designed and annealed into double-stranded DNA identified by sequence. siRNA interference with the four kinds of plasmids (SR-1, SR-2, SR-3, SR-4) were transformed into rat vascular smooth muscle cells to screen the most effective sequence; In order to get the sequence started with interfering carrier by RT-PCR and western-blot, we let the best interfering sequence carriers and pDONR221 to react together. Then entry vectors and lentiviral vectors to express the purpose of pLenti6/V5-DEST response was to restructure the sequence containing interference lentiviral expression vector. Lentiviral vector containing interfering sequence was co-transfected 293T cells to package lentivirus. The virus titer was determined by the expression level of GFP protein in 293T cells. Lentivirus was used to transfect rat bone marrow mesenchymal stem cells.
RESULTS AND CONCLUSION: Sequencing confirmed the successful construction of Nesprin siRNA lentiviral vector LV-siNesprin. The best interference with miRNA plasmid selected by RT-PCR and western-blot was SR-3. The virus titer for lentiviral packaging and concentrating suspension of the activity was 106 TU / ml. The expression of Nesprin protein decreased in transfected cells.By certain conditions, Nesprin-siRNA lentiviral vector can be successfully constructed.

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