Rapid quantitation of recombinant retroviruses.
A method for measuring the concentration of recombinant retrovirus encoding the Escherichia coli LacZ gene is described. The assay is based on the quantitative measurement of beta-galactosidase activity in extracts of cells infected with the LacZ-encoding retrovirus. LacZ-encoded beta-galactosidase activity in transduced cells is measured using the colorimetric substrate, o-nitrophenyl beta-D-galactopyranoside (ONPG), and the results are read using an ELISA plate-reader. Because the entire assay is performed in a 96-well plate, large numbers of samples are easily measured, and the assay has the advantages of rapidity, precision, and ease of data collection. The assay was used to determine the optimal concentration of polybrene, a polycation known to enhance the infectivity of retroviruses, and can be used to evaluate other factors that affect infection, as well as the optimal conditions for production of the recombinant retrovirus.
Morgan
J R
JR
Surgical Services, Massachusetts General Hospital, Boston 02114.
Lee
J
J
Tompkins
R G
RG
Yarmush
M L
ML
D016475:3T3 Cells / Q000201:enzymology / Q000382:microbiology; D000818:Animals; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D007763:Lac Operon; D051379:Mice; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D001616:beta-Galactosidase