CHARACTERIZATION OF A REPLICATION-COMPETENT RETROVIRUS RESULTING FROM RECOMBINATION OF PACKAGING AND VECTOR SEQUENCES

被引:95
作者
OTTO, E [1 ]
JONESTROWER, A [1 ]
VANIN, EF [1 ]
STAMBAUGH, K [1 ]
MUELLER, SN [1 ]
ANDERSON, WF [1 ]
MCGARRITY, GJ [1 ]
机构
[1] UNIV SO CALIF, SCH MED, NORRIS CANC CTR, LOS ANGELES, CA 90033 USA
关键词
D O I
10.1089/hum.1994.5.5-567
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A replication-competent retrovirus (RCR) was detected by S+/L(-) assay in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system.
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页码:567 / 575
页数:9
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