CDNA-transfected cells

Tohda M, Tohda C, Oda H, Nomura Y. Possible involvement of botulinum ADP-ribosyltransferase sensitive low molecular G protein on 5-hydroxytryptamine (5-HT)-induced inositol phosphates formation in 5-HT2c cDNA transfected cells. Neurosci Lett 1995 190 33-36. 

Fogelman SM, Schmider J, Venkatakrishnan K, von Moltice LL, Harmatz JS, Shader RI, Greenblatt DJ. O- and V-dem ethylation of venlafaxine in vitro by human Uver microsomes and by microsomes from cDNA-transfected cells effect of metabolic inhibitors and SSRI antidepressants. Neuropsychopharmacology (1999) 20,480-90. 

FIGURE 5.4 Microphysiometry responses of HEK 293 cells transfected with human calcitonin receptor, (a) Use of microphysiometry to detect receptor expression. Before transfection with human calcitonin receptor cDNA, HEK cells do not respond to human calcitonin. After transfection, calcitonin produces a metabolic response, thereby indicating successful membrane expression of receptors, (b) Cumulative concentration-response curve to human calcitonin shown in real time. Calcitonin added at the arrows in concentrations of 0.01, 0.1, 1.10, and lOOnM. Dose-response curve for the effects seen in panel B. 

It is also important to predict the in vivo biliary excretion clearance in humans, and for this purpose MDCK II cell lines expressing both uptake and efflux transporters may be used (Fig. 12.3) [92, 93]. It has been shown that MRP2 is expressed on the apical membrane, whereas OATP2 and 8 are expressed on the basolateral membrane after cDNA transfection (Fig. 12.3) [92, 93]. The transcellular transport across such double-transfected cells may correspond to the excretion of ligands from blood into bile across hepatocytes. Indeed, the vectorial transport from the basal to apical side was observed for pravastatin only in OATP2- and MRP2-expressing... 

Because the fMet-Leu-Phe receptor is present only at low levels in neutrophils (-12 x 10 15 g of receptor per cell), it has proved difficult to purify and characterise. Researchers have therefore turned to molecular cloning techniques to gain insight into the molecular structure of this receptor. This approach itself has not been easy because, in the absence of an antibody that specifically binds to the receptor, or else without some amino acid sequence data that can be used to synthesise oligonucleotide probes, cDNA libraries cannot be screened to isolate relevant clones. Therefore, experimental systems in which functional fMet-Leu-Phe receptors are expressed on the surfaces of transfected cells have been used. Two main systems have been utilised expression of mRNA injected into Xenopus laevis oocytes and cDNA cloning into the COS-cell expression vector. 

Ishizuka, T., Murata, N., Kanda, T., Kobayashi, I., Ohta, H., Ui, M. and Okajima, F., 1999, Comparison of intrinsic activities of the putative sphingosine 1-phosphate receptor sub-types to regulate several signaling pathways in their cDNA-transfected Chinese hamster ovary cells. J. Biol. Chem. 274 23940-23947. 

Mochizuki, O. N., Nakajima, Y., Nakanishi, S., Ito, S. Characterization of the substance P receptor-mediated calcium influx in cDNA transfected Chinese hamster ovary cells. A possible role of inositol 1,4,5-triphosphate in calcium influx, J. Biol. Chem. 1994, 269, 9651-9658. 

Shi, F.-S., Weber, S., Gan, J.,Rakhmilevich, A.L. andMahvi,D.M. (1999)GM-CSF secreted by cDNA transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF. Cancer Gene Then, 6, 81-88. 

The fourth approach, which eventually proved successful, was expression cloning. In this strategy cDNA from a tissue or cell line expressing the receptor was cloned into a mammalian expression vector and transfected in to a cell line devoid of opioid receptors. Transfected cells could then be screened... 

Krueger S, Haeckel C, Buehling F, Roessner A (1999) Inhibitory effects of antisense cathepsin B cDNA transfection on invasion and motility in a human osteosarcoma cell line. Cancer Res 59 6010-6014... 

Cell and tissue implants Cell suspensions are obtained by trypsinization of confluent cell monolayers. Five microliters containing 2 X 10 cells in medium supplemented with 10% serum are introduced in the corneal micropocket. When the over expression of growth factors by stable transfection of a specific cDNA is studied, one eye is implanted with transfected cells and the other with the wild type cell line. A 0ien tissue samples are tested, samples of 2-3 mg are obtained by cutting the original fragments under sterile conditions. The angiogenic activity of tumor samples is compared with macroscopically healthy tissue. 

Gorelik, E., Xu, R, Henion, T., Anaraki, R and Galili, U. (1997). Reduction of metastatic properties of BL6 melanoma cells expressing terminal fucose(alpha) 1-2-galactose after alpha 1,2-fucosyltransferase cDNA transfection. Cancer Res. 57, 332-336. 

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