Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector.
A retrovirus vector was constructed from the genome of avian erythroblastosis virus ES4. The v-erbA sequences of avian erythroblastosis virus were replaced by those coding for neomycin phosphotransferase, creating a gag-neo fusion protein which provides G418 resistance as a selectable marker. The v-erbB sequences following the splice acceptor were replaced by a cloning linker allowing insertion of foreign genes. The vector has been tested in conjunction with several helper viruses for the transmission of G418 resistance, titer, stability, transcription, and the transduction and expression of foreign genes in both chicken embryo fibroblasts and the QT6 quail cell line. The results show that the vector is capable of producing high titers of Neor virus from stably integrated proviruses. These proviruses express a balanced ratio of genome length to spliced transcripts which are efficiently translated into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells.
Fuerstenberg
S
S
Department of Molecular Biology, Karolinska Institute, Stockholm, Sweden.
Beug
H
H
Introna
M
M
Khazaie
K
K
Muñoz
A
A
Ness
S
S
Nordström
K
K
Sap
J
J
Stanley
I
I
Zenke
M
M
D001355:Alpharetrovirus / Q000235:genetics*; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D011533:Proviruses / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D001616:beta-Galactosidase