The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells

Abstract

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.

FIG. 1.

Modifications made to the MLV vector increase its frequency of expression in F9 EC cells. (A) MLV vector provirus diagram showing the arrangement of elements contained in the full-length MLV vector construct (not drawn to scale). The vector expresses eGFP from the 5′LTR and neomycin resistance from an internal SV40 promoter. Modifications made to the vector are depicted on the left side of the figure. (B) Frequency (± standard deviation) of eGFP expression in unselected F9 EC cells relative to 3T3 cells transduced in parallel. (C) Frequency of eGFP expression in F9 EC cell pools after selection in G418 for expression of Neo^r from a downstream SV40 promoter.

FIG. 2.

The RBS repressed expression in F9 EC cells when positioned downstream of an internal MNDU3 promoter expressing eGFP in a SIN lentiviral vector. (A) Lentiviral vector provirus diagram showing the arrangement of elements in the SIN lentiviral vector series having an internal MNDU3 promoter driving eGFP expression and one of the three PBS sequences inserted between the promoter and the eGFP transgene. (B) Representative flow cytometric analyses of F9 EC and 293 cells transduced in parallel with vectors containing the indicated PBS sequences. (C) Averages (± standard deviation) of flow cytometry data from 15 experiments performed as described for panel B. The percentage of eGFP-positive F9 EC cells was normalized to the percentage of eGFP-positive 293 cells in the same vector arm to control for small differences in vector titer, relative to the F9 EC/293 transduction ratio achieved with vector with no PBS, multiplied by 100. (D) Semiquantitative PCR demonstrates that gene transfer by the vector containing the MLV PBS occurred at relatively the same frequency as for vectors containing the B2, dl587, or no PBS. Mock-transduced cells served as negative controls for eGFP detection by PCR.

FIG. 3.

The RBS repressed expression in F9 EC cells by a lentiviral vector with an internal hUbiqC, hEF-1α, mPGK, SV40, or MNDU3 promoter. (A) Lentiviral vector provirus diagram showing arrangement of elements in the vector series having one of five promoters and either no PBS or an MLV PBS inserted either upstream (←) or downstream (→) of the promoter expressing eGFP. (B) Percentage of eGFP-positive F9 EC cells normalized to the percentage of eGFP-positive 293 cells in the MLV PBS vector arm, relative to the F9 EC/293 transduction ratio achieved with vector with no PBS, multiplied by 100 (± standard deviation).

FIG. 4.

Repression by the RBS is not stem cell specific. (A) eGFP expression in mouse cells from a SIN lentiviral vector with an internal MNDU3 promoter driving eGFP expression with one of the PBS sequences placed between the promoter and the eGFP transgene. Data are presented as the percentage of eGFP-positive target cells (indicated on the x axis) normalized to the percentage of eGFP-positive 293 cells in the same vector arm, relative to the target cell/293 transduction ratio achieved with vector with no PBS, multiplied by 100 (± standard deviation). (B) Semiquantitative PCR demonstrates that gene transfer by the vector containing the MLV PBS occurred at relatively the same frequency as that with vectors containing the B2, dl587, or no PBS.

FIG. 5.

The RBS repressed expression in whole mouse bone marrow and its differentiated progeny after bone marrow transplant. (A) eGFP expression of donor bone marrow kept in culture for 7 days following transduction. (B) Mean eGFP expression (+ standard deviation) in CD45.1⁺ donor cells harvested from recipient mice 10 weeks after transplant with transduced bone marrow shown in panel A. Circles represent values of individual mice. (C) Percentage of donor CD45.1⁺ cells recovered from transplanted mice. (D) Semiquantitative PCR demonstrates that gene transfer by the vector containing the MLV PBS occurred at relatively the same frequency as the B2, dl587, and no PBS vectors in donor bone marrow and bone marrow harvested from each recipient mouse.

FIG. 6.

The RBS repressed expression in the human hematopoietic cell line DU.528 and primary human CD34⁺ CD38⁻ cells isolated from umbilical cord blood. (A) eGFP expression in human cells from a SIN lentiviral vector with an internal MNDU3 promoter driving eGFP expression with one of the PBS sequences placed between the promoter and the eGFP transgene. (B) Semiquantitative PCR demonstrates that gene transfer by the vector containing the MLV PBS occurred at relatively the same frequency as the B2, dl587, and no PBS vectors.

FIG. 7.

EMSA for differentially binding factor A. Differential binding of the factor A bandshift, to an MLV PBS probe but not a B2 PBS probe, was observed in nuclear extracts of several mouse cell lines and primary mouse cells, but not from human DU.528 cells.