Improvement of Retroviral Packaging Cell Lines by Introducing the Polyomavirus Early Region

Abstract

To obtain high-titer recombinant retroviruses, we constructed plasmid pDL+, which carries the extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Although pDL+ is useful for obtaining high-titer recombinant retroviruses, this vector plasmid is difficult to modify further for tissue-specific expression of foreign genes. To overcome this problem, the coding region of the polyomavirus early gene was expressed in the packaging cell lines. We modified the packaging cell lines, psi2 and PA317, by stably introducing the polyomavirus early gene, and established psiMP34 and psiMP37 from psi2, and PAMP51 from PA317. In the transient expression system using plasmids with and without the polyomavirus replication origin, the titers of recombinant retrovirus produced by these cell lines were 10-100 times higher than produced by the parent cell line and reached levels of 0.5-1.5 x 106 cfu/ml. Expression of the polyomavirus early gene in the packaging cell lines did not stimulate the production of replication-competent retrovirus. We also routinely established stable clones producing retrovirus titers over 1 x 10(7) cfu/ml from psiMP34 and PAMP51. We found that the activity of the long terminal repeat (LTR) promoter is stimulated by the polyomavirus early region protein(s) in these cell lines. Therefore, increases titer can be expected to occur in all the retroviral vectors in which LTR promoter is used to transcribe the retroviral genome.

Analysis of the human carcinoembryonic antigen promoter core region in colorectal carcinoma-selective cytosine deaminase gene therapy

“…To improve the efficiency of the retrovirus-mediated gene transfer, several attempts, such as concentration of the retroviruses and modification of the retrovirus-packaging cells, were reported. 18,39,40 In the present study, we established retrovirusproducing clones by the transinfection protocol. The reason for selecting this method rather than direct isolation of vector-transfected producing clones is that the resultant clones generally produce much higher titers of retroviruses and contain only one vector integrant, allowing their structure to be confirmed unambiguously.…”

“…18,19 The retrovirus vector constructs were transfected into the ecotropic retroviruspackaging cell line Psi2 using Lipofectamine reagent (Life Technologies, Grand Island, NY) according to the protocol provided by the manufacturer. Filtered supernatants from ecotropic retrovirus-producing cells were then added to the amphotropic retrovirus-packaging cell line PA317 in the presence of 8 g/mL polybrene (Sigma, St. Louis, Mo).…”

Transduction of glioma cells using a high-titer retroviral vector system and their subsequent migration in brain tumors

“…Replication-competent retroviruses (RCR) were not detected in PAMP51 packaging cells by the marker rescue assay. 11 Plasmid SV/pIP201 + , 11 which contains the neomycin phosphotransferase (neo) gene driven by the 5Ј long terminal repeats (LTR) and the lacZ gene driven by the simian virus 40 (SV40) early promoter, was transfected into PAMP51 cells by using LipofectAMINE Reagent (GIBCO BRL, Gaithersburg, MD, USA) according to the protocol provided by the manufacturer. G418-resistant stable transformants, PAMP51/SV/pIP201 + , were obtained after G418 selection.…”

“…This cell was introduced with polyoma early region. 11 When this vector was transfected into the PAMP51 packaging cell, the supernatant of the cloned retrovirus-producing cells with the highest titer gave 2 × 10 7 c.f.u./ml without the need for any helper virus. A so-called bystander effect has been reported.…”

Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter

“…The retrovirus vector constructs were transfected by using LipofectAMINE Reagent (Gibco BRL, Grand Island, NY, USA) according to the protocol provided by the manufacturers into the ecotropic retrovirus-packaging cell line Psi2, as described previously. 35 Filtered supernatants from ecotropic retrovirus-producing cells were then added to the amphotropic retrovirus-packaging cell line PA317 in the presence of 8 g/ml Polybrene (Sigma Chemical, St Louis, MO, USA). After 48 h, the cells were split and plated in the medium containing 400 g/ml active G418 (Gibco BRL).…”