Mammalian Transfection

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. 

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Also, special vectors allowing expression in both insect cells and mammalian cell cultures from the same vector (pMamaBac [11] andpBacMam [12]) were described, though the amount required for mammalian transfection with one of these vectors is twofold higher than for insect cells, which makes it applicable only for assessment of suitability for a certain cell culture. 

Kain SR, Ganguly S. Uses of fusion genes in mammalian transfection Overview of genetic reporter systems. Curr Protocols Mol Biol 1995 9.6.1-9.6.12. 

Generation of Mammalian Transfectants Expressing Chemokine Receptors 322... 

GENERATION OF MAMMALIAN TRANSFECTANTS EXPRESSING CHEMOKINE RECEPTORS... 

The calcium phosphate method was first used in 1973 to introduce adenovirus DNA into mammalian cells [3]. DNA-Calcium-phosphate complexes are formed by mixing DNA in a phosphate buffer with calcium chloride. These complexes adhere to the cell membrane and enter the cytoplasm by endocytosis. Disadvantages of DEAE-dextran and calcium phosphate transfection are a certain level of cytotoxicity, a complicated transfection procedure, and the fact that not all cell types can be transfected using these methods. 

Factor IX Replacement Hemophilia B therapy may include recombinant (produced via transfection of mammalian cells with the human factor IX gene) or plasma-derived (concentrate from pooled plasma) factor IX (see Table 64-2). Guidelines for choosing the factor-concentrate formulation for hemophilia B are similar to the guidelines for hemophilia A. However, older-generation factor IX concentrates containing other vitamin K-dependent proteins (e.g., factors II, VII, and IX), called prothrombin complex concentrates (PCCs), have been associated with thrombogenic side effects. Consequently, these products are not first-line treatment for hemophilia B.11... 

Geisse, S. and Henke, M. (2005) Large-scale transient transfection of mammalian cells a newly emerging attractive option for recombinant protein production. Journal of Structural and Functional Genomics, 6 (2-3), 165-170. 

Comment It is critical to remove excess cap analogue from in vitro transcribed transcripts prior to transfection, because the cap analogue will compete with transcripts for the cellular translational machinery. Also, even after DNase treatment, the RNA sample may still contain traces of functional pDNA, which may interfere with subsequent detection by RT-PCR. Furthermore, plasmids containing a mammalian promoter may even give rise to de novo transcription in transfected cells. 

Galli, A., Jayanthi, L. D., Ramsey, I. S., Miller, J. W., Fremeau, R. T Jr., and DeFelice, L. J. (1999) L-proline, and L-pipecolate induce enkephalin-sensitive currents in human embryonic kidney 293 cells transfected with the high-affinity mammalian brain L-proline transporter. J. Neurosci. 19, 6290-6297. 

Sitte, H. H., Huck, S Reither, H., Boehm, S., Singer, E. A., and PiU, C. (1998) Carrier-mediated release, transport rates, and charge transfer induced by amphetamine, tyramine, and dopamine in mammalian cells transfected with the human dopamine transporter../. Neu-rochem. 71,1289-1297. 

Transient mammalian High authenticity Relatively fast methods Scale-up difficult Transfection methods cell line-specific... 

With the widespread availability of cell culture facilities, the reduced costs of media and reagents and above all, the commercialisation of a variety of transfection and expression kits, mammalian cells have now become probably the standard for functional studies of transmembrane transporters. Unsurpassed predictivity of the mammalian models may outweigh the higher costs and the lengthiness of the process, compared with bacterial cultures or Xenopus oocytes. Nevertheless, structural studies may require larger amounts than those easily produced in mammalian cells and the appeal of insect cell cultures for... 

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