Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency.
A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells.
Oxford BioMedica (UK) Ltd, Medawar Centre.
J H
JH
Slingsby
D
D
Baban
J
J
Sutton
M
M
Esapa
T
T
Price
S M
SM
Kingsman
A J
AJ
Kingsman
A
A
Slade
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D018014:Gene Transfer Techniques; D015752:Genes, env / Q000235:genetics*; D015750:Genes, gag / Q000235:genetics; D015751:Genes, pol / Q000235:genetics; D006801:Humans; D008979:Moloney murine leukemia virus / Q000235:genetics*; D010710:Phosphates / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011991:Receptors, Virus / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D003470:Culture Media; D010710:Phosphates; D012333:RNA, Messenger; D011991:Receptors, Virus; C067981:leukemia virus receptor, gibbon ape