Targeted insertion of DNA sequences has recently been successful for the first time in cultured mammalian cells. The ability to insert genes exclusively into their natural chromosomal locus could be important for gene therapy if it leads to correct control of their expression.
We aim to evaluate the success of targeted insertion of beta globin sequences into the beta-globin locus in multipotent (MP) hematopoietic stem cells by studying their regulation and function in mice. The current frequency of targeted modification combined with the low concentration of MP stem cells makes the use of whole bone marrow impractical at the present time. However, a means has recently been found to produce murine MP cell lines which have the capacity for indefinite factor dependent self replication in culture, are non-malignant, and can generate cells capable of repopulating the bone marrow of lethally irradiated mice. We will obtain one of these lines, subclone it and verify that our subclone has the characteristics of the parent line. Additional studies to further characterize these lines will include: after rescue of lethally irradiated syngeneic mice having marker chromosomes, determination of how long the donor cells persist and whether or not they remain cytogenetically normal; evaluation of their ability to cure W/W mice; determination that leukemia does not develop in donor cells of the recipient mice; and determination if our stem cell line can repopulate both the lymphoid and myeloid cells of the recipient by marking the donor cells with a DNA sequence introduced into their genome by electroporation. After cells of our subclone are transformed with a plasmid containing a mutant mouse beta globin gene and clones are selected that not only have the mutant beta-globin inserted into the correct position in the chromosomal beta globin locus, but also express the inserted genes in culture, we will test their function in mice as above. Cells with random inserts will be used as controls. If the cells function normally and repopulate the bone marrow, the erythroid and nonerythroid progeny will be tested for appropriate regulation of the inserted gene. Finally, we will test the function of a normal beta-globin gene inserted into the beta globin locus of a new MP stem cell line derived from a beta-thalassemic mouse. Repopulation of the bone marrow and appropriate gene expression will be tested in irradiated beta-thalassemic recipient mice.
| Vecchini, F; Patrene, K D; Boggs, S S (1993) Purified murine hematopoietic stem cells function longer on nonirradiated W41/Wv than on +/+ irradiated stroma. Blood 81:1489-96 |
| Boggs, S S; Boggs, D R; Patrene, K D (1989) The effect of syngeneic marrow injection upon recovery in sub- and near-lethally irradiated mice. Exp Hematol 17:449-54 |
