Improving Identification of the GABAA Receptor in HEK-293 Cells by Using Bicistronic Vectors 

Jaclyn Farber
Marquette University
Mentor: Dr. David Wagner

GABAA receptors, activated by the neurotransmitter ?-aminobutyric acid, are found in every neuron in the brain, and they are inhibitory, meaning that they decrease the likelihood that an action potential will fire. GABAA receptors are heteromeric pentamers, and the one specifically studied here consists of 2?1 and 3?2-GKER subunits. When GABA is applied to these receptors, the ion channel opens up, allowing chloride to flow into the cell, producing an outward current, and hyperpolarizing the cell. 
 In order to study GABAA receptors, we look at electrophysiology recordings taken from outside out patches from human embryonic kidney (HEK-293) cells that have been transfected with DNA constructs of GABAA receptor subunits. Because recording is a tedious process, efficient identification of transfected HEK-293 cells that are expressing GABAA receptors is crucial. To better help us identify cells that are expressing GABAA receptors, the cells are transfected along with Enhanced Green Fluorescent Protein (EGFP).  My goal was to improve the detection efficiency of GABAA expressing cells.  The first major question I am looking at is if the intensity of the EGFP glowing cell corresponds to the amount of current I will get. Although it is the expectation that if EGFP is present in the cell, you should acquire all the DNA, we have found that this is not always reliable using the lipofectamine transfection method.  In the past, pCEP EGFP has been used and about 30% of EGFP expressing cells did not have GABAA current. The correlation between EGFP expression and GABA-evoked current is tested using cells transfected with three different plasmids: pCDNA3.1 human ?1, pCDNA3.1 human ?2-GKER, and pBudCE4.1EGFP.  I recorded from both non-glowing cells and glowing cells, rated subjectively 1-5 on intensity, and saw how much, if any, current is present in each.  I found that 68% of all patches pulled expressing some EGFP had current, and of those patches pulled from cells expressing EGFP with a brightness of at least a 3, 80% had current.  Current size varied greatly in recordings from patches with the same brightness, indicating that EGFP brightness may not be a good indicator of the size of GABA-evoked current. 
It was suspected that the presence of EGFP alone is not the most reliable indicator of GABAA expression.  Therefore, the second major topic I  worked on is devising a new method for better correlation between EGFP glowing cells and GABA-evoked current.  I did this by engineering and transfecting HEK-293 cells with a bicistronic vector in which I can insert two genes onto one plasmid.  I made 3 bicistronic vectors; (1) pBudCE4.1?1/ EGFP (2) pBudCE4.1?2GKER/ EGFP and (3) pBudCE4.1?1/ ?2-GKER.  Using the bicistronic vectors containing EGFP is useful because I know that if EGFP is being expressed, then at least one of the subunits must be present also.
 

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