Gateway-compatible lentiviral transfer vectors for ubiquitin promoter driven expression of fluorescent fusion proteins.
Lentiviral gene delivery has become widely used. Similarly, the Gateway cloning technology that allows restriction-independent cloning of genes into target vectors is becoming increasingly popular. Here, we have generated two Gateway-compatible lentiviral transfer vectors for expression of carboxy-terminal fluorescence tagged fusion proteins, pLVU/GFP and pLVU/RED. We used a restriction enzyme-independent PCR-based approach to introduce the carboxy-terminal fluorescence tags, EmGFP and DsRed, respectively. Both vectors combine the advantages of restriction enzyme/ligation-independent cloning using the Gateway system with a attR1-CmR-ccdB-attR2 recombination cassette, together with expression of fluorescence tagged fusion proteins driven by the robust mammalian ubiquitin C (UbC) promoter. We tested the vectors by expressing different proteins together with the carboxy-terminal fluorescence tags in 293T and SH-SY5Y cells. Both pLVU/GFP and pLVU/RED can be utilized in different experiments, including protein localization studies and live-cell in vivo imaging.
Krupka
Niklas
N
Alzheimer's and Parkinson's Disease Laboratory, Brain and Mind Research Institute, The University of Sydney, 100 Mallett St., Camperdown, NSW 2050, Australia.
Strappe
Padraig
P
Götz
Jürgen
J
Ittner
Lars M
LM
D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D000097573:Red Fluorescent Protein