Co-transfection

FIGURE 5.10 Effects of co-expressed G-protein (G ) on neuropeptide NPY4 receptor responses (NPY-4). (a) Dose-response curves for NPY-4. Ordinates Xenopus laevis melanophore responses (increases light transmission). Ordinates logarithms of molar concentrations of neuropeptide Y peptide agonist PYY. Curves obtained after no co-transfection (labeled 0 jig) and co-transfection with cDNA for Gai6. Numbers next to the curves indicate jig of cDNA of Ga]g used for co-transfection, (b) Maximal response to neuropeptide Y (filled circles) and constitutive activity (open circles) as a function of pg cDNA of co-transfected G g. 

Recombinant assays have revolutionized pharmacology and now functional systems can be constructed with engineered levels of responsiveness (i.e., through difference in receptor levels or co-transfection of other proteins). 

Unmodified PNAs have been introduced to cells by microinjection [33], electroporation [60, 62, 76], co-transfection with partially hybridized DNA oligos [43, 61,... 

A comparative study on the most promising dehvery techniques is presently being undertaken in our laboratory. Based on our experience and the prehminary results from this study, we recommend a protocol based on DNA co-transfection to be the first choice for a delivery protocol ex vivo. 

Co-transfection of synthetic miRs and target plasmids into HeLa cells... 

Co-transfection of an unrelated reference construct is a common practice to compensate for variation in transfection efficiency between cultures. In the systems described here, we use Renilla luciferase as a reporter for miR-mediated repression, and co-transfect Firefly luciferase as a reference for transfection efficiency. Renilla luciferase activity from each transfection is normalized to the corresponding Firefly luciferase measurement. Repression by a miR is then calculated by dividing the normalized Renilla luciferase activity without miR by the normalized Renilla luciferase activity in the presence of miR. 

Figure 6.2 Critical parameters of the miR/mRNA co-transfection method. (A) Titration of mRNA amount. HeLa cells were transfected with increasing amounts of cap tail R-luc-4 sites mRNA and a fixed amount of firefly (F-luc) mRNA. R-luc expression (luciferase activity) was measured 5 h after transfection. (B) Titration of miCXCR4 concentration. HeLa cells were transfected with cap tail R-luc-4 sites mRNA, F-luc mRNA, and varying concentrations of miCXCR4. Luciferase activity was measured 16 h after transfection and fold-repression by the miR was calculated as in Fig. 6.1D (C) Time-course of miR-mediated repression. HeLa cells were co-transfected with cap tail R-luc-4 sites and F-luc (control) mRNAs, either with or without miCXCR4, and harvested at different time points. Repression was calculated as detailed in Fig. 6.1 and plotted against time (mRNA transfection data series depicted by the circles). Analogous plasmid DNA transfections are shown for reference (pDNA, diamonds). Averaged results from several experiments are shown with standard deviation. Data were previously published (Humphreys etal., 2005). Copyright PNAS, reprinted with permission.

Akt activity is induced in a PI-3K-dependent manner immediately suggesting that the phosphorylated lipid products of PI-3K mediate the activation. Incubation of purified Akt with purified 3-phosphorylated phospholipids results in various extents of activation (44,46). These lipids, such as PtdlnsJP, PtdIns(3,4)P2> and PtdIns(3,4,5)P3, specifically associate with PH domains in a number of proteins (47). Furthermore, co-transfection of a dominant-negative form of PI-3K (delta-p85) also inhibits Akt activation (43). It was later shown that introduction of constitutively active mutants of the catalytic subunit of PI-3K was sufficient to activate Akt in cells (46,48). These studies strongly implicate Akt as a downstream effector of growth-factor-stimulated PI-3K activation in a variety of cell types. 

Two of the more recent such approvals are that of Ovitrelle and Luveris. Ovitrelle is the trade name given by Serono to its recombinant hCG-based product. The producer is an engineered CHO cell line that has been co-transfected with the genes coding for both the hCG a- and P-subunits. Downstream processing entails a combination of several chromatographic and ultrafiltration steps and the final product is presented in freeze-dried form. Each vial of product contains 285 fig of active substance (hCG) and the product has been assigned a 2 year shelf-life. It is reconstituted with water for injections (WFI) immediately before use. 

For BmN cells, co-transfection is also performed at 25°C over Sh see Note 13). 

Hepatic peroxisome proliferation depends on a nuclear receptor, PPARa, to mediate these responses in mice, based on lack of response to peroxisome proliferators in PPARa-deficient mice. In one study with another peroxisome proliferator, WY-14,643, carcinogenesis was shown to be dependent on the same receptor. Oral administration of di(2-ethylhexyl) phthalate failed to elicit markers of peroxisome proliferation in PPARa-deficient mice, while the same treatment elicited this response in normal mice. Metabolites of di(2-ethylhexyl) phthalate caused activation of PPARa-mediated gene expression in mammalian cell co-transfection assays. Differences between responsive rodents and humans in various aspects of PPARa-mediated regulation of gene expression are consistent with the lack of activity of di(2-ethylhexyl) phthalate metabolites in hepatocyte cultures from 12 people studied to date. 

Normal p53 protein binds DNA in a sequence-specific manner and thus most likely regulates gene transcription. Co-transfection experiments show that wild-type p53 activates the expression of genes adjacent to a p53 DNA-bind-ing site. Cells bearing oncogenic forms of p53 have lost this activity. 

Table 21.4 Level of gene expression by co-transfection of animals with multiple genes ...

PLL-based carriers have been used as vehicles for DNA vector-based RNAi in combination with a multifunctional envelope-type nanodevice. This combination complexed to a DNA plasmid encoding anti-luciferase siRNA demonstrated 96% inhibition of luciferase activity in an in vitro co-transfection study (57). 

On the other hand, translocation of the MYB transcription factor LCLl fused to pa-GFP from the nucleus to the cytoplasm and its intracellular localization dynamics depends on facilitated nuclear export versus facilitated nuclear import. Protoplasts co-transfected with At2g38360-DsRed and pa-GFP-LCLl were subjected to the 2P-activation procedure and the decrease of nuclear fluorescence intensity was monitored... 

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