Use of Helper Cells with Two Host Ranges to Generate High-Titer Retroviral Vectors

Cosset, François‐Loïc; Girod, Anne; Flamant, Frédéric; Drynda, Antoine; Ronfort, Corinne; Valsesia, Sandrine; Molina, R M; Fauré, Christine; Nigon, Victor; Verdier, Gérard

doi:10.1006/viro.1993.1135

Cited by 19 publications

(13 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Retroviral stocks were assayed for replication-competent virus (RCV) by a vector rescue assay (27) (31)(32)(33). In addition, retrovirus vectors produced from human cells have been shown to be resistant to the mechanisms of virus inactivation involving natural antibodies and complement that occur when virus derived from murine cells is exposed to human serum (2,4).…”

Section: Methodsmentioning

confidence: 99%

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

Ory

Neugeboren

Mulligan

1996

Proc. Natl. Acad. Sci. U.S.A.

859

649

View full text Add to dashboard Cite

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and tet°minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 107 colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of _106 colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (>1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replicationcompetent virus. the VSV-G protein, since the constitutive expression of significant levels of VSV-G in most cells is toxic. However, this method of virus production significantly limits the evaluation of the potential applications of the viral pseudotypes, since only small amounts of virus can be easily produced. To overcome these difficulties, we have generated a stable humanderived cell line which constitutively expresses the necessary retroviral proteins for packaging and provides for large amounts of the VSV-G protein by inducible expression. We describe here the manner in which the cell line was constructed and some of the characteristics of the virus that is generated from the cells. MATERIALS AND METHODSCell Lines and Drug Selections. Adenovirus 5-transformed human embryonic kidney 293 cells (10) were obtained from B. Panning (Whitehead Institute). The 293 cells were grown in 293 growth medium containing Dulbecco's modified eagle medium (DMEM) (GIBCO/BRL), 10% (vol/vol) inactivated fetal bovine serum (IFS) (Sigma), 2 mM L-glutamine (GIBCO/BRL), and 50 units/ml penicillin and streptomycin (GIBCO/BRL). Drug selections in transfected 293 cells were performed at 2 ,ug/ml puromycin (Sigma), 0.3 mg/ml G418 (GIBCO/BRL) and 100 jig/ml Zeocin (Invitrogen). All growth media, except where noted, was supplemented with 1 ,ug/ml tetracycline. NIH 3T3 cells (ATCC CRL 1658) were grown in DMEM containing 10% (vol/vol) calf serum (Sigma), ...

show abstract

Section: Methodsmentioning

confidence: 99%

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

Ory

Neugeboren

Mulligan

1996

Proc. Natl. Acad. Sci. U.S.A.

859

649

View full text Add to dashboard Cite

show abstract

“…Indeed, the duck hepatitis B virus (DHBV), previously produced on human HuH-7 and Hep G2 cell lines with low titers, replicated more efficiently and was produced at higher titer in the avian LMH cell line (Condreay et al, 1990). Moreover, these cells offer the opportunity to develop either helper cell lines able to package defective retroviral vectors (Cosset et al, 1990(Cosset et al, , 1992(Cosset et al, , 1993 or vaccination protocols directed against retroviruses Cosset et al, 1991a).…”

Section: Introductionmentioning

confidence: 98%

Structure and Expression of Endogenous Retroviral Sequences in the Permanent LMH Chicken Cell Line

et al. 1995

Self Cite

View full text Add to dashboard Cite

From DNA mapping data, four endogenous proviral loci have been observed in the chicken permanent cell line LMH. The locus corresponding to endogenous virus (ev) ev1 is present in duplicate whereas the locus corresponding to ev3 is present in one copy. The other loci are probably ev6 and a solitary long terminal repeat. A RNA Northern blot analysis revealed both ev3 and ev6 transcripts but no ev1 transcript was detected. Using avian leukosis virus (ALV)-based vectors, transcomplementing assays were performed. They demonstrate the correct expression and maturation of endogenous env proteins and the absence of production of functional gag and pol components, indicating that these cells are not competent for viral production.

show abstract

“…Ранние упаковочные клеточные линии пред ставляли собой провирус вируса-помощника, в ко тором делетировали упаковочный сигнал. Однако рекомбинации между гомологичными последова тельностями вектора и упаковочного генома как в процессе обратной транскрипции, так и при совме стной упаковке последовательностей вектора и упаковочного мутанта в вирион [12] [42,[46][47][48] в геноме RAV-1 произвели следующие изменения: ^-упако вочный сигнал делетировали, З'-LTR заменили на сигналы полиаделирования; gag-pol-и env-гены экспрессируются в различных плазмидах. Таким образом, для восстановления репликативной ком петентности вируса-помощника необходим не один раунд рекомбинации с последовательностями ре тровирусного вектора, как в случае Q4dh упако вочной клеточной линии, а три раунда рекомбина ции, что маловероятно.…”

unclassified

“…Каждая упаковочная линия продуцирует дан ный Env-белок определенной специфичности и яв ляется устойчивой к инфекции вирусом, «одетым» в эту оболочку [47 ]. Повторные раунды инфекции и амплификация продуцируемого ретровирусного вектора затруднены из-за блокирования клеточных рецепторов, перегруженных £лу-белком.…”

unclassified

“…Совместное культивирование упаковочных клеточных линий с различной хозяйской специ фичностью Isolde (A), Senta (Е) позволило повы сить титр продуцируемого вектора в 10 раз в присутствии репликативно-компетентного вируса, появление которого является результатом рекомби нации между кодирующими последовательностями вируса-помощника, экспрессирующегося в упако вочных клетках, и цис-активными последователь ностями ретровирусного вектора [47 ].…”

unclassified

See 1 more Smart Citation

Usage of retrovirus vectors for gene transfer into birds

Kaida

Rynditch

1997

Biopolym. Cell

View full text Add to dashboard Cite

Use of Helper Cells with Two Host Ranges to Generate High-Titer Retroviral Vectors

Cited by 19 publications

References 0 publications

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

Structure and Expression of Endogenous Retroviral Sequences in the Permanent LMH Chicken Cell Line

Usage of retrovirus vectors for gene transfer into birds

Contact Info

Product

Resources

About