Immortalized rat astrocyte strain genetically modified by rat preprogalanin gene

Abstract

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 μg/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 culture 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.

Keywords

preprogalanin / astrocyte / immortalized / cell strain / biologic minipumps

References

[1] AnK, TianY K, YangH, et al. . Construction of immortalized rat astrocyte strain by transfection of simian virus 40 large T antigen gene. Chin J Anesthesiol, 2004, 24838-838

[2] TatemotoK, RökaeusA, JörwallH, et al. . Galanin—a novel biologically active peptide from porcine intestine. FEBS Lett, 1983, 164124-124

[3] ChngJ L C, ChristofidesN D, AnandP, et al. . Distribution of galanin immunoreactivity in the central nervous system and responses of galanin-containing neuronal pathways to injury. Neuroscience, 1985, 16343-343

[4] LiuH, HökfeltT. Effect of intrathecal galanin and its putative antagonist M35 on pain behavior in a neuropathic pain model. Brain Res, 2000, 88667-67

[5] ZhangX, NicholasA P, HökfeltT. Ultrastructural studies on peptides in the dorsal horn of the spinal cord. I. Coexistence of galanin with other peptides in primary afferents in normal rats. Neuroscience, 1993, 57365-365

[6] KaskK, LangelÜ, BartfaiT. Galanin—a neuropeptide with inhibitory actions. Cell Mol Neurobiol, 1995, 15653-653

[7] LiuH X, BrumovskyP, SchmidtR, et al. . Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: Selective actions via GalR1 and GalR₂ receptors. Pro Natl Acad Sci USA, 2001, 989960-9960

[8] WynickD, ThompsonS W, McMahonS B. The role of galanin as a multi-functional neuropeptide in the nervous system. Curr Opin Pharmacol, 2001, 173-73

[9] SagenJ, PappasG D, PerlowM J. Adrenal medullar transplants in the rat spinal cord reduce pain sensitivity. Brain Res, 1986, 384189-189

[10] EatonM J. Emerging cell and molecular strategies for the study and treatment of painful peripheral neuropathies. J Peripher Nerv Syst, 2000, 559-59