CDNA-transfected cultured cells

Because the amount of a particular receptor expressed is generally quite low (ranging from -2000 to 20,000 molecules per cell), biochemical purification may not be feasible. Genes encoding low-abundance receptors for specific ligands often can be isolated from cDNA libraries transfected into cultured cells. 

In addition, a number of transporters expressed on the basal and apical sides of the proximal and distal tubules are responsible for tubular secretion (Fig. 6.8) (Kusuhara and Sugiyama, 2009) thus, it is necessary to determine the relative contribution of each transporter to elucidate the mechanism for the renal excretion of drugs. For this purpose, an uptake assay using kidney slices and cultured cells transfected with complementary deoxyribonucleic acid (cDNA) of transporters can be conducted (Hasegawa et al., 2003). 

Although we describe the method for adherent tissue culture cells on noncoated chambered cover-glass tissue culture dishes, Lab-Tek chambered cover-glass, the very same protocols also work well with different cell substrates such as MatTek (MatTek, Ashland, MA) culture dishes or glass sUdes. The protocol has been equally successful for transfections of synthetic siRNAs or plasmid cDNAs. 

Specific cDNAs (for example a receptor) inserted in plasmid that transfects a mammalian cell (e.g. fibroblasts) in culture... 

The A. saitoi carboxypeptidase cDNA was cloned downstream of a GDP promoter, and the resulting plasmid, pGCP13, was used to generate recombinant A. saitoi carboxypeptidase protein. No enzymatic activity was detected in the culture supernatant. We detected the A. saitoi carboxypeptidase activity of the extract obtained from yeast cells transfected with A. saitoi carboxypeptidase cDNA in forward orientation (pGCP13), although no activity was observed with the vector alone at pH 3.1. The recombinant A. saitoi carboxypeptidase activity... 

---