Transfection optimization of HEK-293 cells with NMDA receptor subunits NR1 and NR2A Open Access

Taz, Azmain (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/nz805z711?locale=en
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Abstract

Dysregulation of ionotropic N-methyl-D-aspartate receptors (NMDAR) is implicated in numerous brain disorders driving the need for the development of subunit-specific modulators. Cell-based assays are essential for the evaluation of new drugs and thus an optimized protocol for NMDAR expression in cells is important. Here, I investigated the optimal conditions for the transient transfection of HEK-293 cells with Lipofectmine 3000. The effect of different Lipofectamine concentrations, transfection media and cell confluency on transfection efficiency were studied using fluorescence microscopy and western blot. It was found out that DNA:Lipofectamine ratio of 1:2 and 80-90% cell confluency at the time of transfection are needed for high transfection efficiency.

Table of Contents

Table of Contents                                                                                                                     Page

1.      Introduction                                                                                                                          3

2.      Materials and Methods                                                                                                         7 

3.      Results and Discussion                                                                                                      12

4.      Conclusion                                                                                                                         20

5.      References                                                                                                                          22

 

List of Figures

1.      Linear representation of the subunit polypeptide chain and                                                1

schematic illustration of the subunit topology.                                                                    

2.      Schematic diagram of two different transfections.                                                              2

3.      Lipid-mediated gene delivery.                                                                                                          3

4.      pcDNA3.1 constructs of NR1 and NR2A.                                                                                      10

5.      Whole-cell recordings of cells.                                                                                                                  11

6.      GFP images of cells 48 h post-transfection with DNA:Lipofectamine ratio

1:1, 1:1.5, 1:2 and 1:3.                                                                                                                    12

7.       GFP images of cells 48 h post-transfection with DNA:Lipofectamine ratio

1:1.5 and 1:2.                                                                                                                                   13

8.      Fluorescence intensity of GFP from cell lysates on DNA:Lipofectamine

ratio 1:2 transfections.                                                                                                                     14

9.      Fluorescence intensity of GFP from cell lysates on DNA:Lipofectamine

ratio 1:1.5 transfections.                                                                                                                  14

10.   Western blot for NR1.                                                                                                                     15

11.   Western blot for NR2A.                                                                                                                  15

12.  GFP images of cells 48 h post-transfection.                                                                                   16

13.  Brightfield and GFP images of HEK-293 cells 48 h post-transfection.                                         17

 

List of Tables

1.      Concentration and purity of NR1 and NR2A plasmids.                                                    10

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