BacMam Ion Channels
The BacMam System
||Figure 1. This schematic depicts the mechanism of BacMam-mediated gene delivery into a mammalian cell. BacMam particles are pinocytosed and genes are expressed under a specific mammalian promoter. This process begins within 4–6 hours of transduction and is completed after an overnight period in U-2 OS and other cell types.|
Transduction Workflow and Compatible Cells
||Figure 2. Workflow for BacMam-mediated gene delivery into mammalian cells using the extended protocol.
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Convenient Assay Formats
For BacMam K+ channel reagents, we offer the FluxOR™ Potassium Ion Channel Assay Kit to measure ion flux in voltage- and ligand-gated potassium channels with a highly sensitive fluorescent dye.
Figure 3. Dose responses with fresh or frozen U-2 OS cells for two K+ targets in the FluxOR™ Potassium Channel assay. Transduced U-2 OS cells expressing Kv1.3 (panel A) or Kv7.2/Kv7.3 (panel B) were split and plated directly for assay or frozen in liquid nitrogen for one week, thawed, and assayed.
Figure 4. Automated Patch Clamp (APC) measurement of a U-2 OS cell expressing BacMam hERG (Kv11.1) and BacMam Nav1.5 ion channels. U-2 OS cells were co-transduced with 5% vol/vol of each construct as described in the methods. Bottom left panel shows the two step voltage protocol applied to the cells in an Ionworks HT™ instrument. The cells were held at –100 mV and a tail current protocol was applied (blue trace) to measure the outward hERG mediated tail current shown in the left panel. A second step (red trace) was made to 0 mV to capture the peak inward current carried by NaV1.5, shown on an expanded timescale in the right hand panel.
Flexible Expression of Multiple Subunit channels
Figure 5. FluxOR™ potassium ion channel assay signatures of Kv7.2 and Kv 7.3, expressed alone (left panel) or together (right panel). Cells were transduced with 5% vol/vol of each construct alone or together as described in the methods, and subjected to the FluxOR™ assay. A control virus (null BacMam) was used to show the background level of activity in the assay (lowest trace). The stimulus was injected 1:5 vol/vol to yield a final added potassium concentration of 10 mM, and a final added thallium concentration of 2 mM.