Portable cell-based assays using BacMam technology

The BacMam system has been used to create K+ and Na+ ion channel reagents which can be transduced into mammalian cells with ease.  This portability allows for faster assay development in pharmacologically relevant cell types.

The BacMam System

Ion channels are critically important to a number of cellular functions and processes.  One challenge for studying ion channels as drug targets has been in generating robust cellular models.  To address this challenge, we have developed a set of ion channel reagents using the BacMam System.  BacMam technology is based on the use of an insect cell virus (baculovirus) to efficiently deliver and express genes in mammalian cells (Figure 1).
This schematic depicts the mechanism of BacMam-mediated gene delivery into a mammalian cell

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

Cell types successfully transduced with BacMam K+ and Na+ Channel kits include U-2 OS, HEK 293, tSA-201, CHO, HUVEC, and HeLa.  We do not recommend using cells of hematopoietic origin, because they consistently show very poor BacMam transduction.   BacMam K+ and Na+ Channel kits include a virus stock solution (BacMam reagent, Component A) and an enhancer reagent (BacMam enhancer, Component B) for increased expression.  The enhancer is reconstituted in DMSO (Component C) prior to use, and added to cells as indicated in the experimental protocol (Figure 2).

Figure 2. Workflow for BacMam-mediated gene delivery into mammalian cells using the extended protocol.

 Click image to enlarge

Convenient Assay Formats

As a ready-to-use viral stock, BacMam ion channel reagents offer the ability to run assays immediately (i.e. dose response curves or patch clamp experiments) or freeze and store cells after transduction to use at a later date (Figures 4 and 5).  

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.
Dose responses with fresh or frozen U-2 OS cells for two K+ targets in the FluxOR™ Potassium Channel assay
Dose responses with fresh or frozen U-2 OS cells for two K+ targets in the FluxOR™ Potassium Channel assay
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.
Automated Patch Clamp (APC) measurement of a U-2 OS cell expressing BacMam hERG (Kv11.1) and BacMam Nav1.5 ion channels
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

BacMam technology also allows for flexible expression of multiple subunit targets, such as the functional voltage gated potassium channel formed by co-expression of Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3) subunits folded into a tetramer (Figure 5).  Multiple constructs or single species targets can be titrated into the cells for stoichiometry, and optimal expression and signal window in your particular assay.
luxOR™ potassium ion channel assay signatures of Kv7.2 and Kv 7.3, expressed alone (left panel) or together (right panel)

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.

Cannot Find a Reagent You Are Looking For?

Our custom services team can work with you to utilize BacMam technology in your project of interest.  To learn how our experts can get you relevant results faster, email us at discoveryservices@invitrogen.com

For Research Use Only. Not for use in diagnostic procedures.