Transfection efficiency

Chemical modifications of chitosan assayed to enhance cell specificity and transfection efficiency were reviewed. Also, chemical modifications of chitosan were performed to increase the stabihty of chitosan/DNA complexes [95]. 

The urocanic-acid-modified chitosan showed good DNA binding abihty, high protection of DNA from nuclease attack, and low cytotoxicity. The transfection efficiency of chitosan into 293T cells was much enhanced after coup-Hng with urocanic acid [96]. 

Fig. 3. Comparison of transfection efficiencies obtained using PolyFect Reagent, a dendrimer-based transfection reagent, and a calcium phosphate-mediated procedure. COS-7 and HeLa cells were transfected in srx-weU plates with a /3-galactosidase expression plasmid using the appropriate protocol. For the calcium phosphate-mediated transfection, 6 pg of plasmid DNA was used and the medium was changed after 5 h incubation. Transfections were performed in triplicate, and transfection efficiency was measured by monitoring the /3-galactosidase activity of extracts obtained from the transfected cells. The amoimt of /3-galactosidase activity in the extracts correlates with the transfection efficiency. Cells were harvested 48 h post-trans-fection...

The generation number, activation procedure, and core moiety of PolyFect Transfection Reagent have been developed specifically for transfection of certain cell lines, including HeLa and COS-7. Optimized amounts of DNA and den-drimer delivered transfection efficiencies in both cell lines significantly higher than those obtained using a standard transfection method. 

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. 

This chapter will discuss the production and characterization of CLS obtained by different techniques, the formation of the complex between CLS and DNA, and, finally, the in vitro cytotoxicity on different cell lines and the transfection efficiency... 

Emi et al. and Olbrich et al. [19,50] have data about transfection performed with CLS as carriers. For instance, Emi et al. tested the transfection efficiency in primary macrophages and in RAW macrophages. No transfection was observed during 72 h... 

Another important aspect of transfection agents, especially for nonviral systems, was the efficiency/toxicity ratio. The CLS described by Olbrich et al. [19] showed only moderate transfection efficiencies compared with the established polymers... 

Knorr V, Ogris M, Wagner E (2008) An acid sensitive ketal-based polyethylene glycol-oligoethylenimine copolymer mediates improved transfection efficiency at reduced toxicity. Pharm Res 25 2937-2945... 

Peng SF, Yang MJ, Su CJ et al (2009) Effects of incorporation of poly(y-glutamic acid) in chitosan/DNA complex nanoparticles on cellular uptake and transfection efficiency. Biomaterials 30 1797-1808... 

The initial approach to gene therapy involved manipulation of gene expression ex vivo. Toward this end, the desired target cells are identified and subsequently removed from the subject, transfected in vitro, then reintroduced into the patient. A number of protocols have been established for the ex vivo transfection of a wide variety of cell types. This method allows specific cell targeting and high transfection efficiency. However, the process is time consuming, complex, and costly. Additionally, the method is not applicable to all situations, such as those in which an immediate modification is required. 

Once in the cytoplasm, a proportion of plasmid molecules are likely degraded by cytoplasmic nucleases, effectively further reducing transfection efficiencies. There are two potential routes by which plasmid DNA could reach the nucleus ... 

They used different generations of intact dendrimers to transfect plasmid DNA in a variety of cells (Table 18.2) using luciferase, CAT (chloramphenicol acetyltransferase) and /i-galactosidasc reporter genes to quantify transfection efficiency. 

A number of experimental in vivo gene therapy trials on animals using PAMAM dendrimers are in their preliminary stages. Numerous in vivo experiments are currently being conducted in order to optimize the dendrimer generation, concentration, and complex charge ratio to obtain optimal transfection efficiency. 

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