Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

Abstract

BackgroundThe delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected.ResultsStudies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific.ConclusionsIn general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

“…To date, the delivery of foreign DNA into farm animals can be achieved by several methods, including pronuclear microinjection [10], sperm-mediated gene transfer [11,12], electroporation [13], somatic cell nuclear transfer employing genetically modified donor cells [14], and gene transfer mediated by retroviruses [15] or cationic molecules [16]. However, the efficiency of these techniques still remains low.…”

“…In this regard, the use of cationic molecules, such as liposome, polymers, and CPPs, has become a useful tool for gene transfection, due to its low toxicity and relative effectiveness to deliver foreign DNA into somatic cells in vitro [13,16]. These biomaterials are able to readily conjugate nucleic acids and to deliver DNA molecules into a wide variety of mammalian somatic cells [19][20][21].…”

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

“…By manipulating the expression level of particular genes and measuring the differential effect of such manipulations, researchers can deduce the gene functions in the chosen biological systems. Not all transfection methods provide the same transfection efficiencies, and even the same transfection method does not transfect all cell types equally 1 . Hence, different transfection methods have been developed such as calcium phosphate method, DEAE dextran, cationic lipid transfection, cationic non-lipid polymer transfection, electroporation, and nucleofection 2,3 .…”

“…Examples include (1) reporter genes to study the role of different gene elements in gene expression, (2) protein expression plasmids to overexpress the protein of interest, and (3) small interfering RNA to downregulate gene expression. By manipulating the expression level of particular genes and measuring the differential effect of such manipulations, researchers can deduce the gene functions in the chosen biological systems.…”

Application of POLARIC™ fluorophores in an in vivo tumor model

“…However, this requires sophisticated molecular genetic manipulation and laboratory facilities. Furthermore, these techniques are not always adequate for primary cells, which are notoriously difficult to transfect (20). Therefore, DNA transfection is likely to be unsuitable for labeling primary cells soon after harvesting from animals.…”