Transient transfection

FIGURE 3.12 Dependence of constitutive receptor activity as ordinates (expressed as a percent of the maximal response to a full agonist for each receptor) versus magnitude of receptor expression (expressed as the amount of human cDNA used for transient transfection, logarithmic scale) in Xenopus laevis melanophores. Data shown for human chemokine CCR5 receptors (open circles), chemokine CXCR receptors (filled triangles), neuropeptide Y type 1 receptors (filled diamonds), neuropeptide Y type 2 receptors (open squares), and neuropeptide Y type 4 receptors (open inverted triangles). Data recalculated and redrawn from [27],... 

The first idea to consider is the effect of receptor density on sensitivity of a functional system to agonists. Clearly, if quanta of stimulus are delivered to the stimulus-response mechanism of a cell per activated receptor the amount of the total stimulus will be directly proportional to the number of receptors activated. Figure 5.8 shows Gi-protein-mediated responses of melanophores transiently transfected with cDNA for human neuropeptide Y-l receptors. As can be seen from this figure, increasing receptor expression (transfection with increasing concentrations of receptor cDNA) causes an increased potency and maximal response to the neuropeptide Y agonist PYY. 

FIGURE 11.2 Paired experimental data. Values of constitutive calcitonin receptor activity [1 -(Tr/Tj) units] in transiently transfected melanophores. Five separate experiments are shown. Points to the left indicate the basal level of constitutive activity before (filled circles) and after (open circles) addition of 100 nM AC512 (calcitonin receptor inverse agonist). Lines join values for each individual experiment. Points to the right are the mean values for constitutive activity in control (filled circles) and after AC512 (open circles) for all five experiments (bars represent standard errors of the mean). Data shown in Table 11.3. 

FIGURE 11.3 One-way ANOVA (analysis of variance). One-way analysis of variance of basal rates of metabolism in melanophores (as measured by spontaneous dispersion of pigment due to G,.-protein activation) for four experiments. Cells were transiently transfected with cDNA for human calcitonin receptor (8 j-ig/ml) on four separate occasions to induce constitutive receptor activity. The means of the four basal readings for the cells for each experiment (see Table 11.4) are shown in the histogram (with standard errors). The one-way analysis of variance is used to determine whether there is a significant effect of test occasion (any one of the four experiments is different with respect to level of constitutive activity). 

Transiently transfected COS cells Constitutive activity SR 48692 NT Levocabastine... 

Geisse, S. and Henke, M. (2005) Large-scale transient transfection of mammalian cells a newly emerging attractive option for recombinant protein production. Journal of Structural and Functional Genomics, 6 (2-3), 165-170. 

Zhang, L., Schaner, M. E., Giacomini, K. M., Functional characterization of an organic cation transporter (hOCTl) in a transiently transfected human cell line (HeLa),... 

Green fluorescent protein-RhoB GFP-RhoB is localized in endocytic vesicles and has been shown to highlight early endosomes, recycling endosomes, and multivesicular bodies, but is absent from lysosomes (123). Because RhoB is toxic when applied for long periods of time, the cells should be analyzed within 24 hours of transient transfection. 

Eimmel, S., et al.. Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells. Norm. Res., 54, 306-11, 2000. 

Expression by transient transfection methods using mammalian cell lines is a convenient and rapid method of producing recombinant proteins when E. coli systems fail to produce correctly folded, structurally homogeneous protein. Moreover, it is a method that is routinely used to produce proteins for crystallization. 

Durocher, Y., Ferret, S. and Kamen, A. (2002). High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNAl cells. Nucleic Acids Res. 30, E9. 

Pham, P. L., Perret, S., Doan, H. C., Cass, B., St-Laurent, G., Kamen, A. and Durocher, Y. (2003). Large-scale transient transfection of serum-free suspension-growing HEK293 EBNAl cells peptone additives improve cell growth and transfection efficiency. Biotechnol. Bioeng. 84,332-342. 

Pretest these activated shRNA plasmids by transiently transfecting the Maxiprep DNA into ES cells as described in Subheading 3.1.2. 

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