Mammalian Expression Systems and Transfection Techniques
To delineate the function of a single ion channel subtype amongst the multitude that normally constitutes a signalling pathway, it is often insightful to study the function or signalling of that one ion channel in the absence of the others. Mammalian cell
- PDF / 297,456 Bytes
- 12 Pages / 504.57 x 720 pts Page_size
- 59 Downloads / 235 Views
1
Introduction Since many ion channels are regulated by diverse posttranslational modifications (such as phosphorylation, glycosylation, disulfide bond formation) that can differ between species, in order to study mammalian ion channel function in a heterologous system, it is beneficial to use a mammalian expression system. Mammalian cells can be transfected with plasmid DNA encoding ion channel genes. Plasmid DNA may either be introduced into the cells in a transient fashion or may integrate into the cell genome. Cells that have incorporated the plasmid DNA into their genome can be selected via the use of antibiotic resistance genes to generate stable clones that express a protein of interest. This chapter focuses on the methods of transient and stable transfection by lipid-based
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_2, © Springer Science+Business Media, LLC 2013
21
22
Daunia Laurenti and Lezanne Ooi
transfection and transient expression by adenoviral delivery. Three methods of assessing transfection efficiency and identifying transfected cells are also discussed. 1.1 Using Reporters to Analyze the Efficiency of Transfection
The efficiency of transfection is a major concern for many applications and should be monitored using a reporter, such as a fluorescent protein, e.g., enhanced green fluorescent protein (eGFP), to confirm the transfer of nucleic acid to the cell. This can be achieved by (1) transfecting two separate plasmids, one of which encodes eGFP. This is the weakest of the three options presented here; although it is likely that if one plasmid was able to transit through a particular cell membrane then both plasmids were able to do so, it cannot be guaranteed (see Note 1). (2) Generation of a fusion protein, using a plasmid such as pEGFPN1 or pEGFP-C1 (Clontech), in which your protein of interest is tagged with eGFP (as one continuous protein). Careful consideration should be given as to the position of the eGFP relative to the protein of interest (i.e., N-terminal or C-terminal) since the 27 kDa eGFP may affect folding, localization, or function of your target protein. (3) Another option would be to use a bicistronic vector (generates a single mRNA that encodes two proteins), such as pIRES2-eGFP (Clontech). This vector contains an internal ribosomal entry site (IRES) sequence, a nucleotide sequence that promotes translation initiation in the middle of an mRNA and thus allows the simultaneous expression of the protein of interest and eGFP (as a separate protein) from the same mRNA transcript. With this method eGFP expression can be used to determine efficiency of transfection and identify those cells that are positively transfected (since all cells expressing the eGFP were successfully transfected) without the eGFP affecting the folding of the protein of interest. One other important consideration is that the expression of any reporter may affect the expression of other proteins or the function of the cell,
Data Loading...
