Expression of a methotrexate-resistant dihydrofolate reductase gene by transformed hematopoietic cells of mice.

DNA-mediated gene transfer was used to introduce DNA from a methotrexate-resistant mouse fibroblast cell line into mouse bone marrow cells. This cell line contained a methotrexate-resistant dihydrofolate reductase, active at 10(-4) M methotrexate, which was electrophoretically separable from the wild-type mouse enzyme. Transformed hematopoietic cells were returned to irradiated mice and selected in vivo by methotrexate administration. Some recipients of transformed marrow cells expressed the electrophoretically distinct, methotrexate-resistant dihydrofolate reductase in hematopoietic cells. These observations suggest that successful transformation of marrow stem cells to methotrexate resistance is accomplished by insertion of a dihydrofolate reductase gene coding for a mutant enzyme that is highly resistant to methotrexate.

Insertion of new genetic information into bone marrow cells of mice: comparison of two selectable genes.

A system for insertion of new genetic information into mouse hematopoietic cells is described. Two selectable genes were examined: herpesvirus thymidine kinase and a mutant mouse dihydrofolate reductase. The DHFR system appears to be superior in terms of the frequency and stability of gene insertion and expression in hematopoietic tissues. About 70% of mice had indirect (karyotypic) evidence of gene insertion; of these, about 60% (three of five) had stable expression of the inserted mutant DHFR. In contrast, only 13% of mice demonstrated stable karyotypic transformation by HSVtk, and of those with stable transformation five of seven showed persistent viral gene sequences in hematopoietic tissues.

Human ribosomal RNA gene spacer sequences are found interspersed elsewhere in the genome.

A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43-kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to comparative restriction digests. It was estimated that sequences among the five different spacer isolated varied by not more than 1.0%, if all the observed differences are assumed due to point mutation. HaeII-restriction fragments from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.

Insertion of new genes into bone marrow cells of mice.

Drug resistance genes such as those coding for a methotrexate-resistant dihydrofolate reductase (DHFR) or the thymidine kinase from herpes simplex virus can be used to confer a proliferative advantage on bone marrow cells of mice. As a result of this proliferative advantage, transformed cells become the predominant population in the bone marrow. Efficient gene expression was obtained for both the thymidine kinase and DHFR genes inserted into mouse bone marrow. Such gene insertion techniques may ultimately lead to the cure of life-threatening globinopathies such as sickle cell disease or the beta thalassemias. They may also be useful in reducing the hematopoietic toxicity of anticancer drugs.

Insertion of a new gene of viral origin into bone marrow cells of mice.

DNA containing the herpes simplex virus thymidine kinase (HSVtk) gene was used to transform wild-type tk+ mouse L cells to a tk++ status in vitro using methotrexate as a selective agent. HSVtk DNA was also used to transform mouse bone marrow cells in vitro. Transformed marrow cells injected into irradiated and methotrexate-treated recipient mice gave rise to proliferating cells which in some cases dominated the marrow population and which contained HSVtk gene sequences.

Effect of methylcellulose injection on murine hematopoiesis.

This study was designed to determine the effect of methylcellulose (MC)-induced reticuloendothelial (RE) hypertrophy on neutrophils and hematopoietic stem cells and to contrast its overall hematologic effect in the mouse to the more frequently studied rat model. Mice were given MC 3 times/wk and studies were done at 2, 3, and 4 wk, with maximal hematologic change by 2 wk. A stable, but incompletely compensated hemolytic anemia developed which was accompanied by a significant shift of erythropoiesis from marrow to spleen. Thrombocytopenia developed as did neutrophilia, accompanied by an increased number of marrow neutrophil precursors. Extramedullary hematopoiesis was observed in the liver. The number of cells forming spleen colonies in irradiated recipients increased in the spleen but not in marrow. The number of cells producing granulocyte and macrophage colonies in semisolid media increased in spleen and marrow. Splenectomized mice, treated with MC, developed changes very similar to intact mice. Thus, it appears that all three major hematopoietic lines may be destroyed by the MC-hypertrophied RE system. The mouse differs from the rat in its hematologic response to MC by destroying cells in organs other than the spleen, by increasing neutrophil production, by developing hepatic hematopoiesis, and by developing all changes more rapidly.