A simplified general method for determination of recombinant retrovirus titers.
To construct recombinant retroviruses with only a single active promoter, we introduced point mutations into the TATA box region of the 3'-LTR, and successfully obtained high-titer virus with sufficient self-inactivating activity. However, the viral titer could not be determined by the number of G418 resistant colonies since the neomycin resistance gene was under 5'-LTR control, because of inactivation of the selection marker in target glioma cells. To overcome this problem, we constructed PCR primers with homology to a gene under the control of the internal promoter of recombinant retrovirus, and to retrovirus-specific sequences. There was good correlation between the amount of PCR-amplified product and the number of colony forming units when glioma cells were transduced with the retroviruses containing both the neomycin resistance gene and the HTK gene. Amplified PCR products quantitated by densitometry after glioma cells were transduced with SIV retrovirus vectors, and there was good correlation between density and sensitivity to GCV following transduction. Therefore, detection of HTK PCR products from glioma cells transduced with HTK-bearing retroviruses is useful for determining the appropriate packaging cell for efficient production of viral particles. This detection system is especially useful for isolating high titer clones producing SIV-type retroviruses.
Miyao
Y
Y
Department of Neurosurgery, Osaka University Medical School, Japan.
Shimizu
K
K
Tamura
M
M
Yamada
M
M
Tamura
K
K
Nakahira
K
K
Kuriyama
S
S
Hayakawa
T
T
Ikenaka
K
K
D001483:Base Sequence; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012190:Retroviridae / Q000235:genetics / Q000302:isolation & purification*