Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (2): 302-305.doi: 10.3969/j.issn.1673-8225.2010.02.026

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Transfection of mouse L-M (TK-) cells with Wnt3a and its effect on the subcellular distribution of beta-catenin

Shang Yan-chang 1, 2, Wang Shu-hui3, Zhang Cheng2, Xiong Fu2, Li Yong2, Liu Zheng-shan2, Xu Yong-feng2   

  1. 1 Department of Geriatric Neurology, The General Hospital of Chinese PLA, Beijing  100853, China; 2 Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou  510080, Guangdong Province, China; 3 Department of Neurology, Beijing Friendship Hospital, Capital Medical University, Beijing  100050, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Zhang Cheng, Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China; zhangch6@mail.sysu.edu.cn
  • About author:Shang Yan-chang☆, Doctor, Attending physician, Department of Geriatric Neurology, The General Hospital of Chinese PLA, Beijing 100853, China; Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China; yanchangshang@yahoo.com
  • Supported by:

     the National Natural Science Foundation of China, No. 30370510*

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Abstract:

BACKGROUND: Wnt signaling pathway plays an important regulative role in the embryonic development processes. Accordingly, it is of great significance to establish the cell model of Wnt signaling pathway so as to conduct study on it.
OBJECTIVE: To establish Wnt signaling pathway cell model by transfecting L-M (TK-) cells with Wnt3a eukaryotic expression plasmid, and to investigate the effect of canonical Wnt signal pathway on the β-catenin subcellular distribution.
METHODS: The eukaryotic expression plasmid pgk-Wnt3a-pcDNA3.0 after amplification was digested by restriction endonuclease first. Then it was transfected together with the control plasmid pgk-neo-pcDNA3.0 into L-M (TK-) cells via lipofection, after which the cell colony was screened by G418 for amplification. RT-PCR was used for detecting the expression products and the indirect immunofluorescence assay for observing the effect of Wnt3a on the β-catenin subcellular localization of L-M (TK-) cells.
RESULTS AND CONCLUSION: The Wnt3a plasmid was verified by endonuclease digestion to have produced the expected plasmids after amplification. According to the RT-PCR detection to the 10 stably-transfected cell colonies achieved by 3 weeks of G418 screening, it was seen, on the L-Wnt3a cDNA, a strip of bright band of 320 bp in length, which showed that the products of amplification were exactly the expected fragments and that the Wnt3a plasmid was expressed on mRNA transcriptional level after being transfected with L-M (TK-) cells. In contrast, no expected band was found on the cDNA of L-M (TK-) cells transfecting the control plasmid. In addition, the immunofluorescence assay detection showed that the protein expression of Wnt3a was found in the cytoplasm of the L-M(TK-) cells tranfecting Wnt3a plasmid, while for those transfecting the control plasmid, it was opposite. β-catenin, as showing by bright red fluorescence, was found to concentrate and enter into the nucleus of the L-M (TK-) cells transfecting Wnt3a plasmid, while for those transfecting the control plasmid, it was opposite. Cell model with continually activated Wnt signaling pathway is established. The stable expression of Wnt3a in L-M (TK-) cells transfected with pgk-Wnt3a-pcDNA3.0 is obtained. The expression of Wnt3a is able to promote the transfer of β-catenin from cytoplasms into nucleus in L-M (TK-) cells. 

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