Immortalized rat astrocyte strain genetically modified by rat preprogalanin gene.
To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3.1 (+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1 (+)-GAL and pcDNA3.1 (+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 microg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1 (+)-GAL and pcDNA3.1 (+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P< 0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in cultured IAST/GAL were significantly higher than those of IAST and IAST/Neo (P<0.01), but no significant differences were found between the IAST and IAST/Neo (P>0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.
An
Ke
K
Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Tian
Yuke
Y
Yang
Hui
H
Gao
Feng
F
Wang
Peng
P
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D019004:Galanin / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
C056567:GAL protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019004:Galanin