Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells

Abstract

A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-f luouraci-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis.Recombinant retroviruses provide an attractive vehicle for gene transfer based on their capacity for highly efficient infection and nontoxic integration into the genome of a wide range of cell types. Although the potential for retrovirusmediated gene transfer efficiency approaches 100%, this may not be realized because of low viral titers or the failure to stimulate cells to divide, which is required for successful integration. Moreover, expression of the transferred gene(s) may not reach desired levels or be sustained. Such problems are evident in current efforts to apply retroviral gene transfer to the gene therapy of cancer, because if we cannot deliver the antitumor specific drug (1-3) into every tumor cell, then any malignant cell that remains unaffected will emerge as a resistant clone. Recently we have tested the utility of coexpressing the specific antitumor agent and a cDNA encoding a truncated human cell surface antigen CD5 for the immediate postinfection selection of the hematopoietic cells transduced with the retrovirus containing the specific antitumor drug (4). Fluorescence-activated cell sorter (FACS) analysis in combination with functional studies showed that under the conditions used, all cells selected expressed CD5 within 48 hr of termination of the infection procedure. Moreover, the fraction of CD5-positive cells did not decrease with extended propagation of selected cells, indicating that the surface marker lacks deleterious effects on cell growth effect and viability. The use of the truncated CD5 cell surface antigen as a selectable marker of gene transfer offers s...

Stimulation of Mouse Bone Marrow Cells with Kit Ligand, FLT3 Ligand, and Thrombopoietin Leads to Efficient Retrovirus-Mediated Gene Transfer to Stem Cells, Whereas Interleukin 3 and Interleukin 11 Reduce Transduction of Short- and Long-Term Repopulating Cells

“…The results demonstrated loss of EGFP gene expression in individual recipients that were stably reconstituted with MFG-EGFPtransduced cells. Similar silencing of transduced genes in vivo has been observed for other retroviral vectors based on the Moloney murine leukemia virus (Challita and Kohn, 1994;Chen et al, 1997;Garcia-Hernandez et al, 1997).…”

SLUG in cancer development

“…Linear DNA fragments for microinjection were obtained by NotI digestion and injected into CBA Â C57BL/6J fertilized eggs. We identified transgenic mice by Southern analysis of tail snip DNA after EcoRI digestion as described (Garcia-Hernandez et al, 1997). We used the cDNA for mouse Slug for detection of the transgene.…”

Sustained leukaemic phenotype after inactivation of BCR-ABLp190 in mice

“…Linear DNA fragments for microinjection were obtained by NotI digestion and injected into CBA Â C57BL/6J fertilized eggs. We identified transgenic mice by Southern analysis of tail snip DNA after EcoRI digestion as described by Garcia-Hernandez et al (1997). We used a fragment of 863 bp obtained for the human ABL cDNA for the detection of the transgene.…”

“…DNA was digested with restriction endonucleases as described by Garcia-Hernandez et al (1997), separated by electrophoresis in 0.8% agarose and transferred to Hybond-N (Amersham). Filters were UV crosslinked and hybridized to a 32 P-radiolabelled full-length ABL probe.…”