Transfection efficiency cell type

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. 

As an alternative to the chemical transfection systems, the primary cells can be effectively transfected by electroporation (Appendix 5). Amaxa has developed a nucleofection (electroporation) system that is very effective for delivering expression vectors into cells at high efficiency and low cellular mortality. The step-by-step conditions for the transfection of numerous primary cell types, including human airway epithelial cells, can be found at http //www.amaxa.com/primary-cells.html. 

Transfection is the process of introducing DNA or RNA into eukaryotic ceils. The use of transfection is to study the role and regulation of proteins or to understand the mechanisms of a pathway. Transfection can be transient for rapid analysis or stable , mostly for induction of expression. There are various methods of transfection which include electroporation, viral vectors, DEAE-Dextran, calcium phosphate or Lipofectamine. The choice of transfection depends on the cell type used. The most desirable technique is the one which gives high efficiency of nucleic acid transfection with less interference to the cells physiology and high reproducibility. 

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. 

In the two infants a 30% ex vivo transfection efficiency was achieved. Following the re-introduction of the stem cells, each patient s blood was analyzed on a regular basis for receptor expression in appropriate cell types, and for immunological function. After 10 months, the results showed that T- and NK-cell counts were comparable to matched normal infants. Moreover the T and NK cells were found to be completely functional. 

In an original manner, one SV40 NLS sequence has been covalently linked to one end of a linear plasmid encoding Luciferase (Zanta et al., 1999). In combination with polyethylenimine, transfection efficiency was increased 10- to 1000-fold, depending on the cell types compared to the same construction containing a nonfunctional mutated NLS. 

Three families of polymers have been used to study transfection mechanisms polyamines, polyamides, and polyvinyl type polymers. The transfection efficiencies achievable with these systems vary widely, so an in-depth analysis of each polymer family and subsequent comparison of what affects gene delivery will be discussed in this chapter. In addition to high transfection efficiency, it is important for the polymeric systems to be relatively nontoxic to cells in vitro and not to elicit an immune response in vivo. Thus, the effect of transfection parameters on cytotoxicity and immunogenicity will also be examined. 

Fig. 13 Transgene expression efficiency of PGAA polymers in multiple mammalian cell types, (a) G, D, and M polyplexes shown high levels of transfection in multiple cell types. PGAAs with four secondary amines/repeat unit in H9c2 cells in (b) serum-free and (c) serum-containing media. Figures adapted with permissions from [21] and [146], 2005 and 2006 American Chemical Society...

The PAMAM dendrimer-DNA complexes have been shown to promote transfection [152-156]. The earlier studies used reporter luciferase which allows for measurement of luciferase activity in host cells [155, 156]. Although there is some dependence on the cell type being invaded [155], experiments have shown that generations 6-8 dendrimers are most efficient in transfection [155, 156]. There is little transfection with generations 2-4 PAMAM dendrimers and no further improved efficiency with generations 9 and 10 dendrimers. The preferred dendrimer to DNA stoichiometry is 5 1 or higher [155,156]. It has since... 

Some of the critical factors to be considered while choosing the transfection reagent and while designing the experiment are target molecule to be delivered, length of expression, cell type, cellular context, desired viability, desired efficiency, expertise, cost, and time. 

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