Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Abstract

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 x 103 to 5 x 104/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3-to 10-fold by cocultivation of the ecotropic *2 and amphotropic PA-12 helper cells. Higher viral titers (8 x 105 to 7.5 x 106) were obtained when nonproducer NIH 3T3 cells or I2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT-(6-thioguanine resistant [6TGF]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418' could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.

Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources.

“…A band of =3.5 kb is found in amplification with primer TC-65 when hybridized to an HPRT cDNA probe (16). This band was present in amplifications from all hybrids containing the gene but varied in intensity depending upon the amount of human sequence in the hybrid (data not shown).…”

Low-affinity placenta-derived receptors for human granulocyte-macrophage colony-stimulating factor can deliver a proliferative signal to murine hemopoietic cells.

“…The reasons for the relatively high frequency at which the viral genes were turned off are unclear, but this clearly did not depend on viral integration site. Indeed, suppression of retroviral expression has been observed in a variety of different cells, including murine hemopoietic cells (23)(24)(25) and the simian virus 40 early region promoter, used in the present construct to drive the neomycin-resistance gene, has been shown to be particularly sensitive to trans-and cis-acting negative regulatory factors (23)(24)(25)(26)(27).…”

Rapid titration of retroviral vectors encoding intracellular antigens by flow cytometry

“…pZIPTEX DNA (3) was provided by D. Livingston. Hightiter viral stocks were generated by infection or transfection of PA317 cells (15) and repackaging in T-2 cells, as described elsewhere (5,16). G418-resistant T-2 clones were expanded, and medium was collected 24 h after refeeding semiconfluent monolayers.…”