Although plasmids do not naturally exist in mammals, scientists can still reap the benefits of plasmid-based research using synthetic vectors and cultured mammalian cells. Of course, these mammalian vectors must be compatible with the cell type they are tranfected into – a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for example, and a toxin that kills bacteria may not have any discernable effect on mammalian cells. In this blog post we will discuss how mammalian plasmids differ from their bacterial counterparts, including how replication occurs and whether selection is necessary for transfected cells.
Before getting into the mammalian plasmid components, it may be useful to describe the means of introducing genetic material (such as plasmids) into mammalian cells, a process called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction of DNA into bacterial cells); however, the techniques and reagents vary. Plasmid transfection into mammalian cells is fairly straightforward and the resultant cells can either express the plasmid DNA transiently (similar to bacteria) or incorporate the genetic material directly into the genome to form a stable transfection. Unlike bacterial transformation, scientsts do not "select" for cells that have taken up the plasmid in the same way. Selection methods, described below, are typically employed when creating stable cell lines and are not used for general plasmid selection. Instead, reporter genes are often employed to easily monitor transfection efficiencies and expression levels in the cells. Ideally, the chosen reporter is unique to the cell, is expressed from the plasmid, and can be assayed conveniently. A direct test for your gene of interest may be another method to assess transfection success. GFP is often used as a reporter and we will be covering this ever-versatile fluorophore in a later post, so stay tuned!
Transient transfection and the elusive "mammalian ORI"
For many experiments, it is sufficient for the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection process is not integrated into the nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted over time. This, however, may not be a problem depending on the duration or other parameters of your experiment. Mammalian cells double at a much slower rate than that of bacteria (~24 h vs 20 min, respectively). Therefore, it is not always mission critical to make sure the plasmid replicates in the cell, as many of these experiments are concluded within 48 h of transfection.
Of course, it is possible that you may not want the plasmid depleted, but still want to use transient transfection methods. Since there are no "natural" mammalian ORIs, scientists have usurped viral-based ORIs to fill the void. These ORIs, however, require additional components expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen (293E or 293T cells), allow for episomal amplification of plasmids containing the viral EBV or SV40 ORIs, respectively. The presence of these viral components greatly reduces the rate of plasmid dilution but does not guarantee 100% transfection efficiency.
A stable transfection is used to create a population of cells that have fully and successfully incorporated foreign genetic material into their genomes. Unlike plasmids used for expression in yeast and bacteria, plasmids used for stable transfections rarely contain an ORI since the integrated DNA will be replicated as part of the genome. Because the foreign DNA becomes a permanent addition to the host genome, the cells will continually express the genetic traits of the foreign material and will subsequently pass it on to future generations. Stably transfected cells may be considered an entirely new cell line from that of the original parental cells.
Positive selection in mammalian cells
To achieve stable transfection, there should be a selective pressure to force cells to incorporate the plasmid DNA into the genome. For the purposes of this post, we will define positive selection as the means of picking up positive traits (i.e. the plasmid contains a cassette that will make cells resistant to a toxin), whereas negative selection would be the picking up of a negative trait (i.e. the plasmid contains a cassette that will make cells sensitive to a toxin). In the table below we focus on positive selection; however, negative selection techniques can be used in conjunction with positive selection to ensure your gene gets targeted to a specific location within the genome.
Positive selection in mammalian cells works similarly to that in bacteria and a table of the most commonly used selection markers are listed below:
|Name||Gene Conferring Resistance||Cell Types*||Mode of Action**||Working Concentation***|
HeLa, NIH3T3, CHO, COS-1, 293HEK
|Inhibits termination step of translation||2-10 ug/mL|
|G418/Geneticin||neo||HeLa, NIH3T3, CHO, 293HEK, Jurkat T cells||Blocks polypeptide synthesis at 80S; inhibits chain elongation||100-800 ug/mL|
|Hygromycin B||hygB||HeLa, NIH3T3, CHO, Jurkat T cells||Blocks polypeptide synthesis at 80S; inhibits chain elongation.||50-500 ug/mL|
|Puromycin||pac||HeLa, 293HEK, Jurkat T cells||Inhibits protein synthesis; premature chain termination||1-10 ug/mL|
|Zeocin||Sh bla||HeLa, NIH3T3, CHO, COS-1, 293HEK, Jurkat T cells||Complexes with DNA; causes strand scissions||100-400 ug/mL|
*Not comprehensive. ** In eukaryotes. ***The concentration used for selection is typically more (double) than that used for maintenance of a transfected cell line.
Keep these tips in mind:
- There is not one recommended concentration for selection in mammalian cells. Before doing a transfection experiment, it is important to determine the proper concentration required for efficient selection. This is usually achieved by performing a "kill curve" (basically growing cells in various concentrations of the selection reagent). Cells should die within 3-5 days and resistant colonies appear in about 10-14 days depending on how quickly your cells divide.
- Gentamicin is often used as a supplement in mammalian cell culture to suppress bacterial growth, and is not appropriate for mammalian selection – do not confuse this with G418 (aka Geneticin).
- Neomycin should not be used for mammalian expression – instead use G418. This can be confusing since the neo/kan gene confers G418 resistance; however, like gentamicin, neomycin is typically used to suppress bacterial growth.
- The selection agent should not be added to culture media until 24-48 h post transfection when creating stable cell lines.
Looking for Mammalian Expression Plasmids? Check Out Addgene's Empty Backbone Resource:
- Plasmids 101: Antibiotic Resistance Genes
- Plasmids 101: Origin of Replication
- All Plasmids 101 Articles