Reporter plasmid

This assay has been thoroughly reviewed [34] and is outlined in Fig. 5. In brief, a cell is transfected with a reporter plasmid consisting of a GAL4 response element upstream from luciferase. NRs are produced as chimeras consisting of the GAIA... 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

It should be emphasized at this point that the use of physicochemical methods is so far the only way to demonstrate the import of transgene DNA into the mitochondrial matrix in living mammalian cells. The unavailability of a mitochondria-specific reporter plasmid designed for mitochondrial expression severely hampers current efforts toward the development of effective mitochondrial expression vectors. Although any new nonviral transfection system (i.e., cationic lipids, polymers, and others) aimed at the nuclear-cytosolic expression of proteins can be systematically tested and subsequently improved by utilizing anyone of many commercially available reporter gene systems, such a methodical approach to develop mitochondrial transfection systems is currently impossible. 

Gene transfer in vitro and in vivo by cationic lipids is not significantly affected by levels of supercoiling of a reporter plasmid. Pharm. Res. 17 967—973. 

In one report, plasmid DNA (supercoiled Bluescript SK) was digested by Taql restriction enzyme in a Si-glass device with two channels. The flow streams of DNA and enzymes were first introduced in individual channels, and then moved to mix and stopped to react (at 65°C for 10 min) [392]. 

Fig. 3. (A) SK-N-SH neuronal cells were cotransfected with Bcl-xL or Bcl-xL AkB luciferase reporter plasmids together with pSG-c-Rel and pSG-RelA, or a combination ofpSG-p50, pSG-RelA, andpSG-c-Rel expression plasmids. Control cells were transfected with empty pSG5 vector. Twenty-four hours later the luciferase activity was measured. Mutation of NF- B binding site blocked the luciferase expression. The c-Rel-containing dimers, p50/c-Rel and RelA/c-Rel, but not p50/RelA complex, were able to activate Bcl-xL promoter. Values are mean S.E.M. of three experiments run in triplicate ( /> < 0.05 vs. the corresponding control value). (B) OGD-induced NF-acB activation promotes Bim but not Bcl-xL transcription. Primary cortical neurons were transfected with Bim or Bcl-xL luciferase reporter plasmid or with Bim and Bcl-xL AkB luciferase reporter plasmids and then exposed to OGD. Four hours later, luciferase activity was measured. The OGD exposure significantly induced Bim and decreased Bcl-xL promoter activity. Mutation of NF-kB binding sites reduced the luciferase expression. Values are mean S.E.M. of three experiments run in triplicate p < 0.05 vs. the corresponding control value p < 0.05 vs. corresponding wild-type luciferase reporter plasmid). For methods and details, see Samico et al. (2009).

DNA Repair. A connection between p53 and DNA repair was observed in p53-deficient cells that exhibited less global DNA repair [197-199] (but see [200]), as well as a reduced capacity to reactivate cisplatin- and UV-damaged reporter plasmids [173][201 ][202]. Furthermore, pretreatment with low levels of UV activated a protective response in which the levels of repair activity were elevated, an effect not observed in p53-deficient cells [202] [203]. It is possible that the p53 protein is directly involved in removing DNA damage since the protein recognizes both irradiated DNA and mismatches [ 162]. There is also evidence that p53 can interact with several components of the excinuclease, including RPA and the TFIIH-associated factors XPB and XPD [204] [205]. So far, however, there is no evidence to demonstrate a direct role for p53 in the nucleotide excision repair pathway. 

The cyclin-dependent kinase inhibitor p21 is another downstream effector of p53 [161-163], There is evidence for p53-independent induction of p21 [162], and under these conditions the protein may be responsible for cisplatin-induced apoptosis [210][211], The p21 protein usually plays a protective role in response to cisplatin [212] [213], however, an effect which correlated with enhanced repair of a damaged reporter plasmid [212-214], These observations are consistent with the hypothesis that DNA-damage induced p53 activates a G, cell cycle arrest through p21, affording the cell time to repair the lesions and precluding the genetic instability produced by replication of damaged DNA. In accord with this model, the addition of p21 to cell free extracts blocked DNA replication but not excision repair... 

Since the initial paper by Fields and Song, there have been significant technical improvements in the method. DNA-binding domains and transcription activation domains have been optimized to reduce false positives and increase the transcription read-out. A variety of reporter plasmids have been engineered to detect a broad range of protein-protein interactions. Much more is understood about the nature of false positives and how to rout them out. Moreover, in response to the utility of this approach, several laboratories have begun to develop transcription-based assays that can be carried out in bacteria, or protein-protein interaction assays based on alternate readouts such as enzyme complementation or fluorescence resonance energy transfer (FRET). 

Bacterial two-hybrid (B2H) systems have only just begun to be developed. These systems show promise both for transcription repression and activation assays. For these methods to become competitive with the Y2H system, the fusion-protein and reporter plasmids will have to be optimized, just as the original Y2H system was re-... 

Gal4, Spl Utrophin promoter Cell culture (Reporter plasmid) Duchenne muscular (6)... 

G80BP-A 5 -AAGGAGGAGA-3 binding sites In vitro (Reporter plasmid) — (15)... 

Plasmid DNA solution prepare DNA solution e.g. luciferase reporter plasmid or eGFP plasmid, at a concentration of 12 pg DNA/ml by dilution of the stock solution with a serum- and supplement-free medium (e.g., RPMl 1640). 

Luciferase reporter plasmid p55pCMV-lVS-luc +containing the firefly luciferase cDNA under the control of the cytomegalovirus (CMV) promoter. 

To characterize the transfection efficiency in terms of the percentage of transfected cells, prepare the transfection complexes with a eGFP reporter plasmid according to 3.6 and perform transfections according to 3.7. After incubation at 37°C in a... 

ReProGlo assay ReProGlo Luciferase activity (BMC, benchmark concentration) ofTcf/ Lef-promoter-driven reporter plasmid in mouse embryonic stem cells (Wnt signaling) Embryonic development... 

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