Modifications made to the MLV vector increase its frequency of expression in F9 EC cells.
We made a series of retroviral vectors containing from one to three modifications to the MLV vector (5
) to remove cis
-acting elements reported to restrict expression of MLV in F9 EC cells. Figure shows the arrangement of elements contained in the full-length MLV vector constructs (not drawn to scale). The vector expresses eGFP from the 5′LTR and neomycin resistance (Neor
) from an internal SV40 promoter.
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 (more ...)
Figure shows the contribution each of the three modifications, both alone and in combination, made to the frequency of expression in F9 EC cells. To control for differences in titer between vector supernatants, 3T3 cells, which are permissive for MLV expression, were transduced in parallel and under the same conditions as F9 EC cells. Values are expressed as the percentage of eGFP-positive F9 EC cells relative to the percentage of eGFP-positive 3T3 cells 7 days after transduction.
Figure shows that the unmodified MLV vector, L, expresses rarely in F9 EC cells. Replacing the MLV PBS with the dl587 PBS in vector LD, thereby removing the RBS, alleviates repression in a substantial fraction of F9 EC cells. Replacing the MLV U3 with the MPSV U3 alone in vector M, while leaving the MLV PBS and therefore the RBS in place, alleviates very little repression in F9 EC cells. MPSV is a variant of the Moloney sarcoma virus that had greater expression in myeloid cells (34
), and the MPSV LTR was isolated and shown to express better than the MLV LTR in EC cells (11
). Among the seven single-base differences between the MLV and the MPSV enhancer repeats, one of these single-base differences has been shown to introduce a functional Sp1 transcription factor binding site in the MPSV enhancer repeat that is not present in the MLV enhancer repeat. This Sp1 site accounts for much of the increase in activity of this enhancer in F9 EC cells (14
Replacing the MLV U3 with the MPSV U3 and replacing the MLV PBS with the dl587 PBS in combination in vector MD, thereby removing the RBS, alleviates repression in a substantial fraction of F9 EC. Removing the NCR from the U3 of MD to make the triply modified vector MND further alleviates repression in F9 EC cells. In each case where the MLV PBS, and therefore the RBS, is present expression is nearly completely repressed.
Figure shows the contribution that each of these three modifications made to expression in F9 EC cells after selection in G418 for expression of Neor from a downstream internal SV40 promoter. Each of these F9 EC pools was selected with 0.75 μg of G418/ml for 10 days from a pool of cells that was transduced with a dilution of vector supernatant that transduced 3T3 cells in parallel to <10% eGFP positivity, so that most cells within these pools should contain only a single copy of the vector. In 0.75 μg of G418/ml, all nontransduced F9 EC cells in control wells were dead within 7 days. Following G418 selection, the cells were passaged in culture for an additional 2 weeks before analysis of eGFP expression by flow cytometry. Figure demonstrates the same pattern of increased expression of eGFP from the modified vectors as in unselected F9 EC cells. Again, in each case, removing the RBS by replacing the MLV PBS with the dl587 PBS made the largest contribution to increased expression, while changing the enhancer or deleting the NCR made only more modest improvements. Vectors containing the MLV PBS, and therefore the RBS (L and M), expressed in less than 5% of the G418-selected F9 cells, whereas the vectors lacking the RBS (LD, MD, and MND) expressed in 80 to 90% of selected cells.
The RBS repressed expression from an internal MNDU3 promoter in a SIN lentiviral vector.
To determine if the RBS was capable of repressing expression outside of its normal retroviral genome context, a series of SIN lentiviral vectors were constructed having an internal MNDU3 promoter driving eGFP expression with one of the three PBS sequences under investigation inserted between the promoter and the eGFP transgene (Fig. ). SIN lentiviral vectors do not efficiently express transcript from their own LTR after reverse transcription, and thus eGFP expression reflects the activity of the internal promoter (30
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 (more ...)
Each vector supernatant in this series was produced and the titer was determined simultaneously on 293 cells. Based on these titer values, F9 EC and 293 cells were transduced in parallel using identical conditions. After 6 to 10 days the cells were harvested and analyzed for eGFP expression by flow cytometry. Flow cytometric analyses from a typical experiment are shown in Fig. . The averages of 15 experiments are plotted in Fig. . In 293 cells, all four vectors were expressed at the same frequency. In contrast, the lentiviral vector containing the MLV PBS shows essentially no expression in the F9 EC cells, whereas the other vectors, with no PBS or with the B2 or dl587 PBS, all expressed at similar frequencies.
An alternative explanation for the results shown in Fig. is that the vector containing the MLV PBS was not transferred or integrated at the same frequency as the other vectors in the series. To rule out this possibility, we performed semiquantitative PCR on genomic DNA from transduced 293 and F9 EC cells. Figure shows that the vector containing the MLV PBS was transferred at a similar relative frequency as the vectors containing the B2 and dl587 PBS and the vector containing no PBS. Thus, the difference in expression by the vector carrying the MLV PBS is not due to poor gene transfer and therefore reflects repression of expression.
The RBS repressed expression from internal SV40, hUbiqC, hEF-1α, and mPGK promoters.
Previous studies have demonstrated that within the context of the retroviral genome, the RBS was able to repress transcription from two downstream internal heterologous viral promoters, SV40 and AdMLP (31
). To determine whether the RBS also repressed transcription from cellular promoters, we constructed SIN lentiviral vectors analogous to the vector series introduced in Fig. , but having either the SV40, hUbiqC, mPGK, or hEF-1a promoter driving eGFP expression, with and without the MLV PBS sequence inserted either immediately upstream or immediately downstream of the promoter.
F9 EC cells and 293 cells were transduced in parallel with the various vector constructs using identical conditions. Cells were passaged in culture for 6 to 10 days and then analyzed for eGFP expression by flow cytometry. Figure shows the average results from two separate experiments. The data are presented as the percentage of eGFP-positive F9 EC cells normalized to the percentage of eGFP-positive 293 cells in the same vector arm, relative to the F9 EC/293 transduction ratio achieved by vector with no MLV PBS multiplied by 100. Figure shows that the SV40 promoter and the three cellular promoters examined were substantially repressed by the RBS, although to varying degrees. The SV40 promoter was repressed greater than 90% whether the MLV PBS was placed upstream or downstream of the promoter. The hUbiqC promoter was repressed greater than 60%, the hEF-1a promoter was repressed greater than 75%, and the mPGK promoter was repressed greater than 80%. In comparison, the MNDU3 promoter was repressed greater than 95%. These results indicate that although the RBS is capable of substantially repressing heterologous cellular promoters in this context, the repression is not complete when compared to the repression of the MNDU3 promoter.
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 (more ...) Repression by the RBS is more pronounced in undifferentiated stem cells but is not stem cell specific.
Previous studies have described the repressive activity of the RBS as stem cell specific on the basis that restriction is nearly complete in undifferentiated ES and EC cell lines but not in differentiated 3T3 fibroblasts (21
). One study demonstrated that substantial RBS-mediated repressive activity was present in the mouse hematopoietic progenitor cell line FDCP (3
). Previous studies have not examined RBS activity in primary mouse cells. To determine the extent of RBS activity in cell types other than EC and ES cell lines, the series of SIN lentiviral vectors containing the different PBS sequences and the MNDU3 promoter driving eGFP expression (Fig. ) was used to screen for RBS repressive activity in primary mouse cells and mouse cell lines.
The data presented in Fig. demonstrate that RBS-mediated repressive activity is present to varying degrees in mouse cells other than stem cells. Near-complete repression of the vector containing the MLV PBS was seen in F9 EC and ES-D3 cells, as previously reported, and also in WTc.F cells, an ES cell line generated from C57BL/6 mice (37
). In addition, p300 and CBP knockout versions of WTc.F were tested, and similar repression was observed (data not shown).
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. (more ...)
The vector with the MLV PBS was heavily repressed in the three heterogeneous primary mouse cell populations we analyzed. Expression was repressed in greater than 90% of mouse bone marrow stromal cells (CD45−, adherent cells isolated from adult mouse bone marrow), greater than 90% of whole mouse BM cells isolated from adult mice, and greater than 80% of MEFs isolated from 13.5-day embryos of outbred CF-1 mice.
In addition, we observed that expression was repressed to varying degrees in four hematopoietic cell lines at different stages of differentiation: FDCP-Mix cl.A4 (hematopoietic progenitor), AMJ2-cll (macrophage), 70Z/3 (pre-B cell), and BM185 (pre-B cell). Both the 3T3 and STO cell lines showed no substantial repression when compared to the human 293 cell line. Semiquantitative PCR was performed on selected cell types to verify that differences in expression were not attributable to differences in the level of gene transfer (Fig. ). Although the panel of cells examined is limited, these data demonstrate that RBS-mediated repressive activity is not a stem-cell-specific, cell-line-specific, or mouse-strain-specific activity.
The RBS repressed expression in whole mouse bone marrow and its differentiated progeny after bone marrow transplant.
As described above (Fig. ), expression was repressed in greater than 90% of whole mouse BM cells transduced with the SIN lentiviral vector containing the MLV PBS. To determine the fate of the progeny of the bone marrow progenitors in this cell population carrying repressed vector, we transplanted transduced murine BM cells into radiation-ablated mice. Figure shows the percentage of cells expressing eGFP in donor mouse bone marrow transduced for transplantation but maintained in culture for 1 week. Approximately 10 to 12% of the cells expressed eGFP from each vector, except <1% expression was seen in cells transduced by the lentiviral vector containing the MLV PBS. After 10 weeks, the transplanted mice were sacrificed and hematopoietic cells were harvested from the bone marrow, peripheral blood, thymus, and spleen. Figure shows the percentage of donor-derived CD45.1+ cells that expressed eGFP from each mouse in each hematopoietic tissue examined. Expression was nearly completely absent from the vector containing the MLV PBS in CD45+ cells from all four hematopoietic compartments examined. This stands in contrast to the B2 and dl587 PBS vector arms that yielded frequencies of eGFP expression comparable to the vector having no PBS. The absence of eGFP expression in these differentiated hematopoietic cell populations may not reflect active RBS-mediated repression but instead could be due to an inherited epigenetic mechanism of silencing, such as DNA methylation or chromatin condensation, that occurred secondary to RBS-mediated repression in a repopulating hematopoietic progenitor.
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 (more ...)
To rule out the possibility that the lack of eGFP expression observed in the cells transduced with the vector containing the MLV PBS was due to a difference in engraftment efficiency in this arm, harvested cells from all arms were stained with anti-CD45.1 antibody to differentiate donor-derived CD45.1 cells from recipient-derived CD45.2 cells. Figure shows the percentage of harvested cells that were derived from donor cells according to CD45.1 antibody staining. The frequency of CD45.1+ cells recovered from blood, thymus, spleen, and bone marrow of mice transplanted with cells transduced with the MLV PBS-containing vector was in the same range as in the other three vector arms and in the mock-transduced arm. Thus, it can be concluded that the lack of expression seen in the MLV PBS vector arm was not due to inefficient engraftment of donor cells.
To rule out the possibility that the observed lack of eGFP expression seen in the cells transduced by the vector containing the MLV PBS was due to inefficient gene transfer into the donor bone marrow, semiquantitative PCR was performed on genomic DNA isolated from an aliquot of the transduced donor BM cells and bone marrow isolated from each recipient mouse. Donor bone marrow was transduced to the same relative efficiency (Fig. ) in each vector arm. All three bone marrow samples from mice transplanted with bone marrow transduced with the vector containing the MLV PBS were positive for eGFP sequence, and the signal intensity was not significantly different from that seen in the 11 other mice in the three other vector arms. Therefore, the lower expression seen from the vector with the MLV PBS was not due to inefficient gene transfer but rather reflects repression of expression.
The RBS repressed expression in the human hematopoietic cell line DU.528 and primary human CD34+ CD38− cells isolated from umbilical cord blood.
Previous studies have not examined the effects of the RBS in human cells. The series of SIN lentiviral vectors containing the different PBS sequences and the MNDU3 promoter driving eGFP expression (Fig. ) was used to screen for RBS repressive activity in a panel of primary human cells and human cell lines. Of all the cell types we examined, only DU.528 and primary CD34+
cells demonstrated any repressive activity (Fig. ). DU.528 is a hematopoietic progenitor cell line capable of generating progeny with characteristics of at least three hematopoietic lineages, in vitro: T-lymphoid, granulocytic/monocytic, and erythroid (21
cells constitute about 3.5% of CD34+
cells present in umbilical cord blood and are enriched for primitive hematopoietic progenitor cells (17
). About 50% of the cells in each of these cell populations were repressed for expression of the MLV PBS-containing vector. In contrast, little repression was seen in transduced CD34+
cells, which are a heterogeneous population of cells that are more mature than the CD34+
cells. In contrast to mouse EC cells (Fig. ), substantial repression was not observed in the three human EC cells we tested: Tera-2, PA-1, and NCCIT. In addition, we did not observe substantial repression in any of the five human hematopoietic cell lines we tested: U937 (myeloid), KG1a (myeloid), CEM (T cell), Jurkat (T cell), and K562 (myeloid). These findings suggest that expression of a factor(s) that binds to the RBS and represses expression occurs mainly in the more primitive and pluripotent human hematopoietic stem and progenitor cells.
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 (more ...) Binding factor A was present in mouse cells that have RBS-mediated repressive activity.
The differential binding of factor A, from EC cell nuclear extracts, to a MLV PBS probe, but not a B2 PBS probe, has been previously demonstrated by an EMSA (35
). The EMSA gel pictures shown in Fig. demonstrate that the factor A bandshift was present in nuclear extracts from primary mouse cells and mouse cell lines other than EC cells, in each cell type where the MLV PBS was repressive, including cell types where repression by this element was only partial (i.e., BM185 cells).
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. (more ...)
We observed RBS-mediated repressive activity in the human cell line DU.528 and primary CD34+ CD38− cells in our expression assay. To determine if an orthologue of factor A protein was detectable in human cells that showed repression by the MLV PBS, we generated nuclear extracts from DU.528 cells for use in the EMSA. In two experiments, differential binding of any factor to the MLV PBS and B2 PBS probes was not observed. Due to the number of cells required to generate nuclear extracts, we have not attempted this assay with primary human CD34+ CD38− cells.