Abstract | OBJECTIVE: To study the protocol of construction of a KCNQ2-c.812G>T mutant and it's eukaryotic expression vector, the c.812G>T (p.G271V) mutation which was detected in a Chinese pedigree of benign familial infantile convulsions, and to examine the expression of mutant protein in human embyonic kidney (HEK) 293 cells. METHODS: A KCNQ2 mutation c.812G>T was engineered on KCNQ2 cDNAs cloned into pcDNA3.0 by sequence overlap extension PCR and restriction enzymes. HEK293 cells were co-transfected with pRK5-GFP and KCNQ2 plasmid (the wild type or mutant) using lipofectamine and then subjected to confocal microscopy. The transfected cells were immunostained to visualize the intracellular expression of the mutant molecules. RESULTS: Direct sequence analysis revealed a G to T transition at position 812. The c.812G>T mutation was correctly combined to eukaryotic expressive vector pcDNA3.0 and expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both the wild type and mutant molecules on the plasma membrane, which suggested that the c.812G>T mutation at the pore forming region of KCNQ2 channel did not impair normal protein expression in HEK293 cells. CONCLUSIONS: Successful construction of mutant KCNQ2 eukaryotic expression vector and expression of KCNQ2 protein in HEK293 cells provide a basis for further study on the functional effects of convulsion-causing KCNQ2 mutations and for understanding the molecular pathogenesis of epilepsy.
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Authors | Xi-Hui Zhou, Zhi-Yan Hui, Rui-Ming Shi, Hong-Xia Song, Wei Zhang, Li Liu |
Journal | Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics
(Zhongguo Dang Dai Er Ke Za Zhi)
Vol. 13
Issue 8
Pg. 611-6
(Aug 2011)
ISSN: 1008-8830 [Print] China |
PMID | 21849107
(Publication Type: English Abstract, Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- KCNQ2 Potassium Channel
- KCNQ2 protein, human
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Topics |
- Epilepsy, Benign Neonatal
(genetics)
- Fluorescent Antibody Technique
- Genetic Vectors
- HEK293 Cells
- Humans
- Infant, Newborn
- KCNQ2 Potassium Channel
(analysis, genetics, physiology)
- Mutagenesis, Site-Directed
- Polymerase Chain Reaction
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