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Biomedical subjects

B Mintz

Publications and source records attributed to B Mintz.

At least 55 records · Page 3Linked to original sources
PubMed1984

Development of adult bone marrow stem cells in H-2-compatible and -incompatible mouse fetuses.

Bone marrow of normal adult mice was found, after transplacental inoculation, to contain cells still able to seed the livers of early fetuses. The recipients' own hematopoietic stem cells, with a W-mutant defect, were at a selective disadvantage. Progression of donor strain cells to the bone marrow, long-term self-renewal, and differentiation into myeloid and lymphoid derivatives was consistent with the engraftment of totipotent hematopoietic stem cells (THSC) comparable to precursors previously identified (4) in normal fetal liver. More limited stem cells, specific for the myeloid or lymphoid cell lineages, were not detected in adult bone marrow. The bone marrow THSC, however, had a generally lower capacity for self-renewal than did fetal liver THSC. They had also embarked upon irreversible changes in gene expression, including partial histocompatibility restriction. While completely allogeneic fetal liver THSC were readily accepted by fetuses, H-2 incompatibility only occasionally resulted in engraftment of adult bone marrow cells and, in these cases, was often associated with sudden death at 3-5 mo. On the other hand, H-2 compatibility, even with histocompatibility differences at other loci, was sufficient to ensure long-term success as often as with fetal liver THSC.

Aging↗
PubMed1984

Monoclonal derivation of mouse myeloid and lymphoid lineages from totipotent hematopoietic stem cells experimentally engrafted in fetal hosts.

Mutant mouse fetuses with a hematopoietic stem cell defect were injected with a mixture of two normal strains of fetal liver cells to test the possibility of seeding with single stem cells and of deriving all hematopoietic lineages clonally. Recipients were either Wf/Wf, with a mild endogenous defect offering only marginal selective advantage to a normal donor cell, or W/W, with a severe defect. Among 11 Wf/Wf animals with long-term grafts, 8 had only one or the other of the donor strains. Some of these individuals must have been seeded by only a single donor cell (P = 0.1); the frequency of this event was at least 20% (90% confidence) and most likely 50% of the cases. Cell-specific strain markers in myeloid and lymphoid lineages reinforced the likelihood that renewal and differentiation had occurred from a totipotent hematopoietic stem cell. In a smaller W/W group, some hosts were seeded by at most two cells (P = 0.1), and single-cell seeding could not be ruled out. The experiment allows stem cell pedigrees to be examined during the normal developmental progression. In both groups observed here, some mice displayed a regular and complementary rise and fall in proportions of cells of different genotypes, thereby suggesting clonal succession in a hierarchy of stem cell compartments. This transplant system also offers advantages for future experiments on regulated expression in vivo of genes transferred (in vitro) into totipotent hematopoietic stem cells.

Animals↗
PubMed1983

Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germ line.

The mutagenic potential of recombinant DNA in the germ line was investigated in the descendants of mice obtained from eggs injected with the human growth-hormone gene in a pBR322 vector. Six positive animals (including one mosaic) were produced and had 1-20 copies of the donor sequences in unique and often complex integration patterns indicative of a single or an interrupted insertion. All were heterozygotes and transmitted the foreign insert to their progeny, forming six new HUGH strains. Matings between heterozygotes yielded viable healthy homozygotes in four of the strains. However, in the HUGH/3 and HUGH/4 strains, no postnatal homozygotes were found and litter sizes at birth were small. These two independent cases of mutation, both homozygous recessive prenatal lethals, are attributable to disruption of native sequences by alien ones. They constitute the first instances of insertional mutagenesis due to integration of recombinant DNA in the germ line of the mouse. The mutants provide new possibilities for molecular identification of gene functions necessary for normal development.

Animals↗
PubMed1982

Human beta-globin gene sequences injected into mouse eggs, retained in adults, and transmitted to progeny.

Foreign gene sequences were retained in two adult mice (out of 62 analyzed) from fertilized eggs injected with a recombinant plasmid containing the human beta-globin genomic region and the herpes simplex viral thymidine kinase gene. The intact human and viral genes were found in DNA of one of the animals and, in the other, at least part of the human globin gene was present. The latter individual transmitted these sequences to its progeny in a Mendelian ration. Thus, human DNA may be incorporated into the germ line of mice for in vivo studies of regulation of gene expression in development, genetic diseases, and malignancy.

Animals↗
PubMed1982

Totipotent hematopoietic stem cells: normal self-renewal and differentiation after transplantation between mouse fetuses.

Successful engraftment of mouse fetal liver cells in early fetal recipients, after microinjection via the placental circulation, is attributable to seeding of the recipient's liver by a cell type that is ancestral to both the myeloid and lymphoid definitive lineages and is capable of sustained self-renewal and differentiation for more than 2 years. This primitive cell is therefore the normal totipotent hematopoietic stem cell (THSC). The use of a large series of mutant anemic recipients with decreasing severity of an endogenous stem-cell defect (W/W, Wv/Wv, Wf/Wf, Wv/+), and therefore of graded selective advantage to normal donor cells, has revealed that engraftment entails marginal numbers of cells--probably individual ones--in the least afflicted hosts. Thus the observed progressive and coordinate shift toward donor-strain erythrocytes, granulocytes and B and T lymphocytes, over time, indicates THSC expansion to form a larger stem-cell pool and normally regulated differentiation of cells from the pool. This transplant system allows allogeneic combinations with impunity and therefore provides many novel experimental possibilities for investigating THSC normal development, genetic abnormalities or neoplastic potential in relation to the intact developmental succession of hematopoietic tissue environments in vivo.

Anemia↗
PubMed1982

Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human beta-globin gene.

Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, we investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH beta 1 containing the human genomic beta-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human beta-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.

Animals↗
PubMed1981

METT-1: a karyotypically normal in vitro line of developmentally totipotent mouse teratocarcinoma cells.

A karyotypically normal, chromosomally female (X/X) in vitro line of mouse teratocarcinoma stem cells was established from a malignant mouse teratocarcinoma of the 129/Sv Sl C P inbred strain. The tumor of origin was experimentally induced by ectopic transplantation of a 6-day embryo. The normal number of chromosomes was observed in 92% of metaphases of the cultured cells. This high frequency of euploidy, as well as karyotypic normalcy, were maintained during numerous passages in culture without a feeder-cell layer and after freezing and thawing of the cells. The line has been designated METT-1 (Mouse Euploid Totipotent Teratocarcinoma), signifying that it is the first such in vitro line that has proved (in tests by T. Stewart and B. Mintz, manuscript in preparation) to be developmentally totipotent, i.e., capable of both somatic and germinal differentiation when injected into blastocysts, even after freezing and thawing and prolonged culture. This unique ensemble of properties renders the cell line suitable for selection of specific mutant genes and for gene-transfer experiments in culture, for the purpose of producing from the mutant cells new strains of mice with predetermined genetic changes.

Animals↗
PubMed1981

Successive generations of mice produced from an established culture line of euploid teratocarcinoma cells.

The possibility of utilizing mouse teratocarcinoma stem cells as intermediaries for production of new strains of mice with preselected mutant or foreign genes requires that, after propagation in culture (to allow for genetic manipulation and selection), the cells be capable of normalization and orderly development in carrier embryos and, ultimately, of germ-cell formation. Heretofore, no in vitro cell line has fulfilled all these requirements. A karyotypically normal teratocarcinoma culture line was recently established in this laboratory and now has been investigated as a candidate. The line, designated METT-1, is chromosomally female (X/X) and was obtained from the 129 (agouti-colored) inbred strain [Mintz, B. & Cronmiller, C. (1981) Somat Cell Genet 7, 489-505]. The developmental potential of these cells was tested, after prolonged culture and freezing and thawing, by microinjecting them into early (blastocyst stage) embryos of the C57BL/6 (black) strain. Among 312 experimental animals examined at 1 week of age, there were 41 mice (21 females and 20 males) that displayed the coat colors of both strains. This frequency (13%), as well as the extent of the coat areas derived from the cell line, greatly surpasses the contributions observed in all previous experiments, whether with other in vitro teratocarcinoma cell lines or with in vivo transplant lines. The developmental totipotency of METT-1 cells became evident from the presence of substantial amounts of 129-strain cells (bearing an isozyme marker) in all internal tissues of an individual whose coat was largely agouti. The culture-cell lineage also proved to be capable of giving rise to reproductively functional oocytes. Of nine mosaic-coat females testmated to C57BL/6 males, one produced progeny of the diagnostic agouti color in two litters; these heterozygous F(1) offspring in turn transmitted their marker genes to F(2) homozygous segregants. Thus, the METT-1 teratocarcinoma line bridges the gap between in vitro cell propagation and in vivo development and between the soma and the germ line. This creates the option of producing new mouse strains with predetermined genetic changes designed as probes of developmental regulation or as models of human genetic diseases.

Alleles↗
PubMed1981

The human beta-globin gene and a functional viral thymidine kinase gene in developing mice.

Two foreign cloned genes--one encoding a tissue-specific protein and one encoding a constitutive enzyme--were introduced into mouse eggs by microinjection into a pronucleus shortly after fertilization. They were the adult human genomic beta-globin gene and the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of herpes simplex virus (HSV), ligated in the pBR322 plasmid. Thirty-three developing mice were autopsied in late fetal life; all appeared normal. Blot hybridization tests revealed that the DNA of as many as five (15%) of the fetuses (from separate litters), and of their corresponding placentas, contained copies of the human beta-globin gene and of the HSV tk gene that had been retained and replicated without significant loss or rearrangement. The estimated total numbers of copies per cell were 3-50 for the donor globin gene and 3-20 for the donor tk. In some of the fetuses, these totals included some copies of molecular weight higher than that of the intact sequence; the additional segments may have arisen through changes such as deletions or duplications. The foreign genes in the five positive fetuses appear to be present in high molecular weight DNA. Assays capable of distinguishing between foreign and native TK indicated that at least one of the fetuses with the HSV tk gene had some TK enzyme of the HSV type and, therefore, at least one gene copy that was being accurately transcribed and translated to produce a functional protein, despite the absence of selective pressure. Thus, pure recombinant genes introduced into mice at the onset of their development can remain intact and be stably incorporated and even expressed. These experiments provide a practical basis for novel investigations of the developmental control of normal gene expression in vivo of the causes and possible cures of genetic diseases.

Animals↗
PubMed1980

Introduction of a viral thymidine kinase gene and the human beta-globin gene into developmentally multipotential mouse teratocarcinoma cells.

Teratocarcinoma (TCC) stem cells provide unique prospects for the introduction of specific genes into mice, by virtue of their dual capacity for propagation in vitro and for normal differentiation in embryos. In this study, we have demonstrated that foreign genes amenable to selection in culture can be transferred into the stem cells and expressed. These cells maintain expression of the gene for long periods during differentiation in tumors in vivo in the absence of selective pressure. The cells also integrate an unlinked nonselectable gene at high frequency. Addition of the cloned herpes simplex virus (HSV) thymidine kinase (tk; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene to cultures of tk(-)TCC cells yielded tk(+) colonies at a frequency of one colony per 4 mug of plasmid DNA. This transformation efficiency, although appreciably lower than for mouse L tk(-) cells, permits the isolation of many transformants. The HSV provenance of the transformed phenotype was verified by the characteristic electrophoretic mobility of the tk protein and by neutralization of the tk activity with specific antiserum. Moreover, blot hybridization tests revealed at least one intact copy of the viral tk gene integrated into the DNA of transformed cells. When injected into syngeneic mice, the cells formed solid tumors with various differentiating tissues. From blot hybridization comparisons with their cell lines of origin, seven of nine tumors examined had maintained the HSV tk gene without significant loss or rearrangement. Viral tk enzyme activity could also be demonstrated in at least some of the tumors. Cotransfer of the cloned human beta-globin gene along with the unlinked HSV tk gene was successful in 2 of 10 tk(+) transformants. Thus, defined genes can be stably introduced into TCC cells in culture and maintained in vivo in a form in which they are transcribed and translated to produce a functional protein.

Animals↗
PubMed1979

Mouse teratocarcinoma mutant clones deficient in adenine phosphoribosyltransferase and developmentally pluripotent.

Mouse teratocarcinoma stem cells deficient in activity of adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) were obtained in order to have this marker in developmentally versatile cells. Mutagenized stem-cell cultures were selected for resistance to 8-azaadenine and four clonal cell lines were isolated. Three had severe deficiencies of APRT activity (7% or less of wild type) and one had a moderate reduction (73%). The enzyme in the latter clone was found to be an electrophoretic variant with slightly less anodal migration than the wild-type enzyme. Each clone remained stably APRT-deficient for at least 3 1/2 weeks, after subcutaneous inoculation, in the absence of the selective agent. The tumors formed from the inocula comprised a variety of differentiated tissues and thus showed persistence of stem-cell developmental pluripotency despite mutagenesis and selection. All mutants also retained the quasinormal karyotype (X/O sex chromosomal constitution, trisomy-19) of the parent line. These lines are appropriate for such uses as production (by blastocyst injection) of mouse models of the human genetic deficiency and for foreign-gene transfer, via teratocarcinoma cells, into mice.

Adenine Phosphoribosyltransferase↗