title: Stable cell line of human SH-SY5Y uniformly expressing TWIK-related acid-sensitive potassium channel and eGFP fusion.
abstract: TWIK-related acid-sensitive potassium channels (TASK3) are pharmacological targets of CNS inflammation induced by acidification. They function as molecular switches between survival and death of neurons. In this report, TASK3 cloned from human brain cDNA was tagged with enhanced green fluorescent protein (eGFP), and the fusion gene was transiently expressed in human neuroblastoma SH-SY5Y cells. A cell line stably expressing TASK-eGFP fusion proteins was generated from transient expression cells by using fluorescence-activated cell sorting followed by antibiotic selection. The uniform expression of TASK3 fusion proteins was further confirmed by flow cytometry. Moreover, the localization of TASK3 tagged with eGFP was checked by confocal microcopy. TASK3-eGFP fusion proteins are observed on the SH-SY5Y cell membrane. The strategies using eGFP as a fusion tag facilitate the monitoring of the TASK3 expression and enable the successful employment of FACS for screening and construction of cell lines stably expressing TASK3. The TASK3 overexpression cell line will lay a fundamental for the in vitro evaluation of TASK3 function during hypoxic/ischemic injury.
authors: Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, Henan, 453003, People's Republic of China, lichaokun@hotmail.com.
authors: Chaokun
authors: C
authors: Li
authors: Linyu
authors: L
authors: Wei
authors: Hongbo
authors: H
authors: Jiang
authors: Linlin
authors: L
authors: Shan
authors: Xinjuan
authors: X
authors: Li
authors: Na
authors: N
authors: Lu
authors: Guohong
authors: G
authors: Wang
authors: Dongliang
authors: D
authors: Li
mesh_terms: D001923:Brain Chemistry; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D018613:Microscopy, Confocal; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D024683:Potassium Channels, Tandem Pore Domain / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; C498655:KCNK9 protein, human; D024683:Potassium Channels, Tandem Pore Domain; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins