Establishment of HEK293 cell lines stably expressing human parathyroid hormone receptors.
- Author:
Yue MENG
1
;
Miaomiao XIE
;
Zhen LIN
;
Liang YUAN
;
Wei LI
;
Song HAO
;
Dehong YANG
Author Information
- Publication Type:Journal Article
- MeSH: Gene Expression; Genetic Vectors; HEK293 Cells; Humans; Plasmids; Receptors, Parathyroid Hormone; genetics; metabolism; Sequence Analysis, DNA; Signal Transduction; genetics; Transfection
- From: Journal of Southern Medical University 2013;33(7):956-961
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo establish HEK293 cell lines with stable expression of human parathyroid hormone (PTH) receptors.
METHODSThe purified gene fragments of PTH-related peptide receptor (PTHR) and its mutant form (DSEL) were cloned separately into pcDNA3.1(+) vector after digestion with EcoR I and Not I, and the resulted pcDNA3.1(+)-PTHR and pcDNA3.1(+)-DSEL plasmids were verified by restriction enzyme digestion and DNA sequencing. HEK293 cells were transfected with these plasmids and the expression of PTHR and DSEL in the cells were examined by RT-PCR and ELSIA.
RESULTSSequencing and restriction enzyme digestion analysis showed that PTHR and DSEL cDNAs were correctly cloned into pcDNA3.1(+)vector. After a 48-h transfection of HEK293 cells with the recombinant plasmids and G418 selection, the positive cell clones stably expressing the constructs were obtained, which showed expressions of PTHR and DSEL mRNAs detected by RT-PCR. These positive cells showed high levels of PLC and aAMP production in response to PTH stimulation.
CONCLUSIONThe HEK293 cell lines with stable expression of PTH1R or DSEL gene established in this study provide useful cell models for studying the physiological functions of PTH peptides.
