Transfection assay

Baert, L., Wobus, C. E., Van Coillie, E., Thackray, L. B., Debevere, J., and Uyttendaele, M. (2008c). Detection of murine norovirus 1 by using plaque assay, transfection assay, and real-time reverse transcription-PCR before and after heat exposure. Appl. Environ. Microbiol. 74, 543-546. 

Oct-1 and 2 138 Yes. POU homeodomain interacts with Increased transcription in vivo (transfection assay)... 

RAG1,2 72 Yes (transfection assay) and association (co-immunoprecipation) in vivo. Activation requires basic region of SSRPl Increased cleavage in vitro... 

Fig. 1. Fligti-ttiroughput RNAi screening using siRNA libraries. Assay plates containing library siRNA are treated with (1) diluted transfection reagent to complex the siRNA and lipid. (2) Cells are added to initiate the transfections. Assay plates are incubated for 72-96 h then (3) readout reagent is added. Assay plates are read and the data is analyzed (4-5).

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. 

The frequency of active ras oncogenes in human bladder cancer (F6) associated with schistosomiasis was examined. Of nine squamous cell carcinomas of the bladder, none scored as positive in the regular DNA transfection assay. The restriction fragment polymorphism assay at codon 12 of the H-ras gene confirmed the absence of an activating mutation at this site in all samples. Western blotting analyses of the ras p21 proteins suggested a point mutation with codon 61 in one sample only. Enhanced expression of the ras p21 protein was demonstrated in four samples. 

These structural analyses are complemented by transfection analyses from the same irradiated samples. In each experiment the amount of transfection was reduced by radiation exposure to a level of 1 to 9% of the control. Further exposure did not reduce this level. The transfection assay has a background value due to intrinsic cell fluorescence which is independent of radiation damage. Similar phenomena have been reported in irradiation of enzymes in which there was a small non-enzymatic reaction producing the same product. As in those cases, this component could be subtracted from all samples, giving a simple exponential decrease in transfection activity (Fig. 3). In three experiments the target size associated with transfection activity was 1067 +/-155 kDa, considerably smaller than the 3300 kDa total mass (Table I). 

A detailed functional analysis of the immunoglobulin promoter elements is not available. However, comparison of sequences from a number of immunoglobulin promoters revealed the presence of a well-conserved octanucleotide consensus (ATGCAAAT) in all VH promoters and its complement (ATTTGCAT) in all VL promoters as well as in the heavy chain enhancer [54,55], This octanucleotide has been shown to be essential for transcription in both VH and VK promoters, as deletion of it abolishes promoter activity [46,55,56]. Furthermore, in the presence of the IgH enhancer, the octanucleotide is a sufficient VH upstream promoter element as measured in transfection assays [57],... 

Apart from the octanucleotide no other strongly conserved consensus sequences have been identified in VH gene promoters, though this does not exclude the presence of other c/s-acting elements. A second consensus is found in the promoter of VK genes [55] this sequence (TGCAGCGTG) is also found in the heavy chain enhancer. However, this element is less well conserved than the octamer and mutations within it do not appear to alter the activity of a VK promoter in transfection assays [52,56], No nuclear factor has yet been found which binds this element, and its significance remains unclear. 

Once a transcription factor is isolated and purified, its partial amino acid sequence can be determined and used to clone the gene or cDNA encoding it, as outlined in Chapter 9. The isolated gene can then be used to test the ability of the encoded protein to activate or repress transcription in an in vivo transfection assay (Figure 11-16). 

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