Avian retroviral expression of luciferase.
Biologically active replication-competent (subgroups A, B, and C) and replication-defective Rous sarcoma virus-derived vectors containing the cDNA encoding firefly luciferase as a reporter gene were constructed. In these retroviral vectors, luciferase is expressed from a spliced subgenomic mRNA. A biologically active replication-defective UR2 virus-derived vector expressing the reporter gene as a gag-luciferase fusion protein from an unspliced genomic mRNA was also constructed. The luciferase reporter gene was used because it lacks homology with chicken genomic sequences and because a rapid and sensitive direct enzymatic assay is available to monitor luciferase expression in retrovirus-infected cells. The levels of luciferase expression in luciferase recombinant retrovirus-infected chicken embryo fibroblasts are greater than 10(3) higher than that detected in uninfected cells or in cells infected with retroviral vectors carrying other genes. Endpoint dilution titration experiments demonstrated that one infected cell can be detected in a background of 10(3) uninfected cells. The vectors are stable in tissue culture and high level expression of the unselected luciferase reporter gene is maintained. The vectors were used to express luciferase in chicken embryos, demonstrating the potential utility of luciferase as a reporter in vivo.
Garber
E A
EA
Department of Animal Biochemistry and Molecular Biology, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
Rosenblum
C I
CI
Chute
H T
HT
Scheidel
L M
LM
Chen
H
H
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D015870:Gene Expression / Q000235:genetics*; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000235:genetics
D015683:Gene Products, gag; D011993:Recombinant Fusion Proteins; D008156:Luciferases