Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (28): 5313-5316.doi: 10.3969/j.issn.1673-8225.2010.28.044

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Construction and identification of mouse SK2 gene subclones

Zhang Qian1, Yuan Jian-dang1, Zhao Guo-qiang2, An Yu-hui3   

  1. 1 Department of Physiology, 2 Department of Immunology, 3 Department of Biochemistry and Molecular Biology, College of Basic Medicine, Zhengzhou University, Zhengzhou   450052, Henan Province, China
  • Online:2010-07-09 Published:2010-07-09
  • About author:Zhang Qian☆, Doctor, Professor, Doctoral supervisor, Department of Physiology, College of Basic Medicine, Zhengzhou University, Zhengzhou 450052, Henan Province, China qianzhang@zzu.edu.cn
  • Supported by:

    the National Natural Science Foundation of China, No. 30870909*

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Abstract:

BACKGROUND: Small conductance, Ca2+-activated potassium (SK) channel, presents in various cell types and plays a crucial role in action potential profile. However, coupling and modulation of calcium and associated molecules to SK2 channel remains unclear.
OBJECTIVE: To construct the recombinants of pGBAT7 and target fragments of SK2 gene, so as to observe the coupling and regulation of SK2 channel gene to calcium and other molecules.
METHODS: Three pairs of primers of the target fragments of SK2 gene were designed and synthesized based on the full-length sequences of SK2. After being identified, they were individually sub-cloned into the yeast expressive plasmid pGBKT7 to construct pGBKT7-SK2 vectors. The recombinant pGBKT7-SK2 vectors were transformed into yeast AH109 by electroporation, and their activation was tested. The recombinants were extracted from yeast AH109 and verified by electrophoresis and sequencing.
RESULTS AND CONCLUSION: The target fragments of SK2 gene by PCR were 411, 546 and 729 bp, respectively. Three sub-clones of pGBKT7-SK2 were successfully constructed. Electrophoresis and sequencing showed that the constructed sub-clones of pGBKT7-SK2 met the expected requirements. The recombinant pGBKT7-SK2 vectors transformed into the yeast could be activated. The successful construction of the sub-clones of SK2 gene provides an important material basis for further study in the SK2 channel and function-associated molecules.

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