Fusion Gene Vectors Allowing for Simultaneous Drug Selection, Cell Labeling, and Reporter Assay in Vitro and in Vivo

• Published in: Analytical chemistry (Washington) Vol. 84; no. 2; pp. 987 - 993
• Main Authors: Zhao, Haobin, Hong, Ni, Lu, Wenqing, Zeng, Huaqiang, Song, Jianxing, Hong, Yunhan
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 17.01.2012
• Subjects: Acetyltransferases - genetics; Analytical chemistry; Animals; Animals, Genetically Modified; Cells, Cultured; Chemistry; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Exact sciences and technology; Fish; Fluorescence in situ hybridization; Gene expression; Gene Fusion; Genetic Vectors; Germ Cells - cytology; Germ Cells - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; In Situ Hybridization, Fluorescence; In Vitro Techniques; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Oryzias - genetics; Oryzias - metabolism; Plasmids - genetics; Promoter Regions, Genetic; Protein Synthesis Inhibitors - pharmacology; Puromycin - pharmacology; Red Fluorescent Protein; RNA Helicases - genetics; Stem cells; Transgenes - physiology; Fold; Chemical analysis; Selection; Cell biology; Testicle; Betaherpesvirinae; Human cytomegalovirus; Chemical enrichment; Eye; Transfection; Gene; Efficiency; Pisces; Fluorescence in situ hybridization; Regulatory sequence; Fusion protein; Vector; Quantitative analysis; Clone; Drug; Herpesviridae; In vitro; Genetic transfer; Virus; Plasmid; In vivo; Vertebrata; Green fluorescent protein
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac202541t

Vector systems allowing simultaneously for rapid drug selection, cell labeling, and reporter assay are highly desirable in biomedical research including stem cell biology. Here, we present such a vector system including pCVpf or pCVpr, plasmids that express pf or pr, a fusion protein between puromycin acetyltransferase and green or red fluorescent protein from CV, the human cytomegalovirus enhancer/promoter. Transfection with pCVpf or pCVpr produced a ∼10% efficiency of gene transfer. A 2-day pulse puromycin selection resulted in ∼13-fold enrichment for transgenic cells, and continuous puromycin selection produced stable transgenic stem cell clones with retained pluripotency. Furthermore, we developed a PAC assay protocol for quantification of transgene expression. To test the usefulness for cell labeling and PAC assay in vivo, we constructed pVASpf containing pf linked to the regulatory sequence of medaka germ gene vasa and generated transgenic fish with visible GFP expression in germ cells. PAC assay revealed the highest expression in the testis. Interestingly, PAC activity was also detectable in somatic organs including the eye, which was validated by fluorescence in situ hybridization. Therefore, the pf and pr vectors provide a useful system for simultaneous drug selection, live labeling, and reporter assay in vitro and in vivo.