5 Reasons why BacMam is replacing plasmid transfection

BacMam is a BSL-1 vector for efficient transduction in most mammalian cells, including primary cultures, cell lines, and iPSC derived cells. For several years, researchers have used BacMam to express the biosensors developed at Montana Molecular in their cells of interest. In response to their requests, we have developed process to package other genes in BacMam for a low cost reliable alternative to transient transfection. Here we’ll explore the benefits of BacMam and understand why many researchers are making the switch.

Are you stuck in a tranfection rut?

 
Are your transfection reagents holding you back?
 
Do you need a more powerful approach to transfection?
 
Here are 5 reasons why researchers are using BacMam to overcome challenges of transient transfection:
Reason #1 Significant Cost Savings

Reason #1

Significant Cost Savings

Transfection reagents: $.10-$.25+/well

Custom BacMam production: ∼$.06/well

Reason #2 Improve work flow

Reduce media exchange steps.

Less liquid handling is better.

Reason # 3 Reduce cell-to-cell variation in expression

Figure 1A illustrates the response of anindividual HEK 293T cell expressing an Upward DAG sensor in an imaging experiment. The addition of a G-protein-coupled receptor (GPCR) agonist that signals through Gq causes an increase (∆F) in the fluorescence of the cells over time. The background is defined as the fluorescence produced by the plate, media, and cells alone. One can see in this response that the overall brightness of the cell sets the difference between the autofluorescence and the sensor signal: the brighter the cell, the better the signal-to-noise ratio will be. Another key parameter, however, is the absolute change in fluorescence, ∆F. One can imagine a dim sensor with a large ∆F being better than a bright sensor with a small ∆F. The experiment illustrated in Figure 1 was done by transiently transfecting HEK 293T cells using plasmids and Lipofectamine 2000. Plasmid transfection produces a broad range of fluorescence intensities in the cells: com-
parison of a few cells in any well reveals that there is more than a 10-fold variation in intensity (Fig. 1B). This variation exceeds the dynamic range of the camera and has several consequences. First, this saturation in an imaging instrument means that the responses of the brightest cellsare lost, and they become another source of background fluorescence that lowers the apparent ∆F. Second, very high levels of sensor expression can exceed the amount of analyte within the cell, resulting in less bound sensor and less change in fluorescence, ∆F. The variability inherent in transient transfections can be overcome with a variety of heterologous expression strategies that introduce a consistent amount of the sensor gene.The creation of stable cell lines or transgenic animals mini-mizes expression variability, but this is a limited solution since many investigators already have a particular cell line of choice, or they study a different species. Viral delivery, on the other hand, provides a flexible solution that is compatible with many different cell types. Baculovirus is par-ticularly useful because it can be used to drive sensor expression in mammalian cells, if the correct promoters areused, while the remaining viral genome is silent.15 The virus concentration can be easily adjusted, and it carries minimal risks to humans, making it easily used in automated laboratory settings.

Remarkably consistent expression of the Upward DAG2 sensor16 was achieved within wells and across plates using a baculovirus expression system . This consistency reduced the variability, significantly improving Z′from 0.59 in an optimal transient transfection to 0.72 in abaculovirus transduction. Our efforts to improve Z′ by increasing ∆F, however, brought ever diminishing returns. We realized that the solution might be to increase the signal-to-noise ratio by making brighter sensors.

Reason #4: Efficient expression in more cell types

including iPSC-derived and primary cultures

Significant Cost Savings

Transfection reagents: $.10-$.25+/well

Custom BacMam production: ∼$.06/well

Reason #5: Optimize expression levels

Reason #1

Significant Cost Savings

Transfection reagents: $.10-$.25+/well

Custom BacMam production: ∼$.06/well