Team:Hong Kong HKUST/characterization/cmv

Introduction

CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.

In our characterization, CMV promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108). The pCMV-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope. The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator (BBa_K648013) that does not contain the CMV promoter. The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.

Method

CMV promoter was characterized by obtaining in vivo expression of green fluorescence protein in mammalian cell. To do so, we have built a CMV characterization construct by fusing CMV promoter with green fluorescence protein (GFP) and hGH polyadenylation sequence (hGH pA) in pSB1C3 backbone. The construct was transfected in HEK293FT cells along with GFP – pSB1C3 construct for negative control and pEGFP-N1 for positive control. We have cloned CMV promoter out from pEGFP-N1 (Addgene), which contains CMV promoter and EGFP reporter. The green fluorescence was observed under confocal microscope.

Characterization Procedure

1. Build:

- CMV characterization construct: CMV promoter – Green Fluorescence Protein (GFP) – hGH polyadenylation sequence (hGH pA) - pSB1C3 (BBa_K1119006 – BBa_K648013 – BBa_K404108 – pSB1C3)

- Negative control construct: GFP – hGH pA - pSB1C3 (BBa_K648013 – BBa_K404108 – pSB1C3)

2. Culture HEK293FT cell line (see below)

3. Transfect CMV characterization (CMV – GFP – hGH pA – pSB1C3), negative control (GFP – hGH pA – pSB1C3) and positive control (pEGFP-N1) plasmids in HEK293FT cell line. *We cloned CMV promoter out from pEGFP-N1 (Addgene) that contains CMV promoter and enhanced green fluorescence protein (EGFP). We used this plasmid as our positive control for CMV promoter characterization.

4. Observe GFP signal under confocal microscope

Cell Culture and Transfection

We cultured HEK 293FT cells following ATCC’s standard procedure, except that we have used DMEM with 10% FBS and 1% penicillin/streptomycin for culture medium. For transfection, we have followed the manufacturer’s protocol of LipofectamineTM 2000 (Invitrogen). We have used serum free and antibiotics free DMEM to form DNA-lipofectamine complex.

Result

Figure 1. CMV promoter drives expression of GFP. HEK cells transfected with pCMV-GFP gave GFP signals. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals. Scale bar = 10 microns

Conclusion

Reference