Transfection optimization

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. 

When assayed in HEK293 cells transfected with the cloned human, rat and guinea pig TRPVl, (23a) showed similar potencies. Not unexpeetedly, (23a) showed poor metabolic stability and a structure-activity study to optimize potency and drug-like properties was initiated. Modification on the left-handed A -aryl section showed that ... 

Genetic methods, in multiobjective optimization, 26 1033 Genetics, of yeast, 26 480 481 Genetic selection, 12 452 Genetic software techniques, 10 342 Gene transfection, dendrimers in, 26 791-792... 

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. 

Saravolac EG, Ludkovski O, Skirrow R, et al. Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity. J Drug Target 2000 7 423. 

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).

Liposomes can be modified in numerous fashions by the addition of peptide sequences. The benefit of this is that the inclusion of the peptide allows the lipoplex to be optimized for its task such as the targeting a specific cell type with a specific receptor ligand sequence or the transfection of nondividing cells, with an NLS. 

The optimal weight ratio of LAH4-L1 to siRNA for transfection was determined previously as 10 1. A range of different ratios should be tried for other peptides if the optimal ratio has not been determined. 

To optimize the amount of siRNA for in vitro transfection, prepare the pep tide/siRNA complexes with various concentrations of siRNA, ranging from 1 pmol to 100 pmol per ml. The optimal amount of siRNA required for efficient gene silencing effects varies between the cell types used. 

Depending on the cell division rate, optimal gene silencing effects may be observed at different time points post transfection. It is suggested to perform western blotting at 48-96 h post transfection to determine the optimal time point. 

It is recommended to linearize the plasmid before transfection to avoid aberrant integration of Tet-repressor coding sequence. Alternatively the construct can be delivered by viral-mediated infection. In this case use pLenti6/TRto express the repressor. It is recommended to optimize the transfection conditions by using an easily detectable reporter system such as green fluorescent protein (GFP). 

Be sure to know the transfection efficiency. Poorly transfected cells could generate false-negative results. Set up a priori the optimal condition of transfection with a reporter gene. 

While the initial goal of the discovery process is to express and purify the recombinant protein, without regard to cost and as quickly as possible, ultimately the production of a recombinant protein must be optimized to ensure that it can be produced at a reasonable cost. In most cases expression systems used to produce small quantities of recombinant protein for preliminary evaluation are not suitable to produce large quantities. Therefore, the gene inserted in the initial expression vectors must be systematically modified and recloned into vectors that can produce (1) a high yield of expressed protein, (2) stable host cell transfections, and (3) protein excretion. 

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