Team:Hong Kong HKUST/characterization/cmv

Introduction

CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.

For this promoter's characterization we assembled it with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108). The P_cmv-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. The negative control was the same as the experimental construct, but minus the promoter. The detailed protocols employed for our characterization work can be accessed via the link.

Characterization Procedure

1. Build:

- CMV promoter 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 promoter 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 HEK293FT cells following American Type Culture Collection’s standard procedure, except that we used DMEM with 10% FBS and 1% penicillin/streptomycin in our culture medium. For transfection, we followed the manufacturer’s protocol of LipofectamineTM 2000 (Invitrogen) and used serum-free and antibiotics-free DMEM to form the DNA-lipofectamine complex.

Result

Figure 1. CMV promoter drives expression of GFP. HEK293FT cells transfected with P_cmv-GFP gave GFP signals. HEK293FT 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 = 10µm.

Conclusion

Reference