University of Kentucky
The Fusion of Luciferase and Green Flourescent Proteins in Transgenic Mouse Models
Institution
University of Kentucky
Faculty Advisor/ Mentor
Brett Spear
Abstract
Our understanding of gene regulation in living organisms has been greatly facilitated by the use of reporter genes. Two of the most commonly used reporter genes are the jellyfish gene coding for green fluorescent protein (GFP) and the firefly gene coding for luciferase (LUC). Future mouse experiments that our lab would like to pursue require the expression of both GFP and LUC in the same mouse. Therefore, my project has been to generate a fusion reporter protein that contains both GFP and LUC activity. Using PCR and recombinant DNA technologies, I have linked GFP and LUC in the same reading frame, and cloned this hybrid gene downstream of the cytomegalovirus promoter. Testing of this new fusion protein was conducted by transient transfections into Hela cells. Results indicated that LUC levels were very high; however, GFP expression was lower than expected. To increase GFP expression, we added a viral sequence leading to ribosome skipping. The new sequence was amplified by PCR, sequenced, and inserted between GFP and LUC. This construct was again tested. The positive control vectors for GFP and LUC were EGFP-C1 and pGL3, respectively. LUC levels are still high in the new vector, and we observed a substantial improvement in GFP levels. This data shows that the viral ribosome-skipping element leads to increased GFP protein levels. We are currently generating transgenic mice containing the GFP-LUC gene. Analysis of GFP and LUC expression will be performed to determine whether these mice will be useful for subsequent experiments in the lab.
The Fusion of Luciferase and Green Flourescent Proteins in Transgenic Mouse Models
Our understanding of gene regulation in living organisms has been greatly facilitated by the use of reporter genes. Two of the most commonly used reporter genes are the jellyfish gene coding for green fluorescent protein (GFP) and the firefly gene coding for luciferase (LUC). Future mouse experiments that our lab would like to pursue require the expression of both GFP and LUC in the same mouse. Therefore, my project has been to generate a fusion reporter protein that contains both GFP and LUC activity. Using PCR and recombinant DNA technologies, I have linked GFP and LUC in the same reading frame, and cloned this hybrid gene downstream of the cytomegalovirus promoter. Testing of this new fusion protein was conducted by transient transfections into Hela cells. Results indicated that LUC levels were very high; however, GFP expression was lower than expected. To increase GFP expression, we added a viral sequence leading to ribosome skipping. The new sequence was amplified by PCR, sequenced, and inserted between GFP and LUC. This construct was again tested. The positive control vectors for GFP and LUC were EGFP-C1 and pGL3, respectively. LUC levels are still high in the new vector, and we observed a substantial improvement in GFP levels. This data shows that the viral ribosome-skipping element leads to increased GFP protein levels. We are currently generating transgenic mice containing the GFP-LUC gene. Analysis of GFP and LUC expression will be performed to determine whether these mice will be useful for subsequent experiments in the lab.