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M A Eglitis

Publications and source records attributed to M A Eglitis.

34 records · Page 2Linked to original sources
PubMed1988

Retroviral-mediated gene transfer into hemopoietic cells.

Retroviral vectors have provided a means for the introduction of functioning exogenous genes into the hematopoietic system of whole animals. Although these vectors are quite efficient in the mouse model, when applied to non-murine in vivo systems, the efficiency of gene transfer has diminished to impractical levels. Since in vivo analyses are expensive and time consuming, in vitro models have been developed to speed the evaluation of alternative protocols. Using in vitro colony assays, three approaches were evaluated for their ability to improve the infectivity of hematopoietic progenitor cells with retroviral vectors. Exogenously applied hematopoietic growth factors increased the proportion of hematopoietic colonies in vitro up to an average of 5 fold. When alternative sources of progenitors, such as fetal cord blood, were used, improvements in infection efficiency were also obtained. Finally, evidence was acquired suggesting that xenotropic packaging of vectors also improved infection efficiency.

Animals↗
PubMed1988

Gene transfer into hematopoietic progenitor cells from normal and cyclic hematopoietic dogs using retroviral vectors.

The Moloney murine leukemia retrovirus-derived vector N2 was used to transfer the bacterial NeoR gene (conferring resistance to the neomycin analogue G418) into hematopoietic progenitor cells. Approximately 5% of day seven CFU-GM were resistant to 2,000 micrograms/ml G418, using a supernatant infection protocol in the absence of vector-producing cells. A greater proportion of CFU-GM colonies were recovered relative to uninfected controls as the stringency of selection was diminished. Enzyme activity was detected in drug-resistant colonies, confirming that the resistant colonies obtained after infection with N2 represented cells producing neomycin phosphotransferase. Activity in the CFU-GM colonies approached 50% of that of drug-resistant vector-producing cells on a per cell basis. To test the hypothesis that more rapidly cycling bone marrow cells would be more susceptible to vector infection, we treated progenitor cells obtained from cyclic hematopoietic (CH) dogs with the N2 vector. Despite the increased numbers of hematopoietic progenitor cells obtained from CH dogs, the proportion of G418-resistant CFU-GM did not increase over that obtained with N2-infected normal marrow. These results demonstrate that retroviral vectors can be used to transfer and express exogenous genes in canine hematopoietic progenitor cells.

Animals↗
PubMed1987

Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.

Primate bone marrow cells were infected with a retroviral vector carrying the genes for human adenosine deaminase (h-ADA) and bacterial neomycin resistance (neor). The infected cells were infused back into the lethally irradiated donor animals. Several monkeys fully reconstituted and were shown to express the h-ADA and neor genes at low levels in their recirculating hematopoietic cells for short periods of time.

Adenosine Deaminase↗
PubMed1987

Gene therapy: efforts at developing large animal models for autologous bone marrow transplant and gene transfer with retroviral vectors.

Two new large animal models, non-human primates and fetal sheep, have been developed in an effort to determine the feasibility of using retroviruses for gene therapy. The retroviral vectors N2 and SAX have been used to introduce the genes for neomycin phosphotransferase (neoR, conferring resistance to the antibiotic G418) and human adenosine deaminase (ADA; EC 3.5.4.17), respectively. Varying levels of human ADA activity have been detected in six of the eight SAX-treated monkeys analysed. In the monkey with the greatest activity, human ADA levels approximately 0.5% of endogenous monkey ADA levels were detected. By in situ hybridization, roughly one in 100 bone marrow cells were found to express vector DNA. Sheep have been used for studies of the infectability of fetal blood progenitors in vivo. Blood cells were treated with the N2 vector at the 96th day of gestation, and marrow cells were assayed for the presence of G418-resistant haematopoietic progenitors, starting from one week after birth (62 days after treatment). Up to 33% of colony-forming progenitors were drug resistant initially and, although the proportion of resistant colony-forming units declined, a level of 10% has been found 153 days after transplantation. Human bone marrow has also been treated with the N2 vector, resulting in 1-2% G418-resistant progenitors.

Animals↗
PubMed1987

Retroviral-mediated gene transfer into mammalian cells.

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.

Adenosine Deaminase↗
PubMed1986

Correction of adenosine deaminase deficiency in cultured human T and B cells by retrovirus-mediated gene transfer.

A retroviral vector called SAX, containing the cloned human cDNA for adenosine deaminase (ADA), has been constructed and used to introduce the ADA gene into cultured T- and B-lymphocyte lines derived from patients with ADA deficiency. DNA analysis showed that the SAX vector was inserted intact into the T and B cells at approximately one copy per cell. The treated cells produced the characteristic isozymes of human ADA at a level similar to normal T and B lymphocytes. It is known that ADA-deficient lymphocytes are unusually sensitive to high levels of 2'-deoxyadenosine, and this is the mechanism thought to underlie the selective lymphocytotoxicity associated with ADA deficiency in vivo. Expression of the introduced ADA gene was sufficient to reverse the hypersensitivity of these genetically deficient lymphocytes to 2'-deoxyadenosine toxicity. These results support the suggestion that retroviral vector gene-delivery systems show promise for application to human gene therapy.

Adenosine Deaminase↗
PubMed1986

Reversible and irreversible effects of retinol upon the phenotypic properties of embryonal carcinoma cells.

We have generated an embryonal carcinoma cell line, NR1, which is not growth-inhibited in response to retinol. Although the retinol-treated cells undergo morphological change, show reduced levels of the surface antigen SSEA-1 and possess increased surface reactivity with antifibronectin serum, the extent of phenotypic change of NR1 cells in response to retinol is not so great as that following treatment with retinoic acid. Furthermore, unlike cells from lines such as F9, the retinol-promoted morphological alterations appear to be reversible. The increased adherence of NR1 cells to glass coverslips in the presence of retinol suggests that stronger interaction with the substratum is responsible for the observed alteration in appearance of the cells. It is possible that NR1 cells exposed to retinol progress no further than a reversible, early stage of differentiation. Alternatively, retinol-treated cells might express a group of markers normally associated with, or accompanying, an irreversible, differentiated phenotype even though the cells are not, in fact, undergoing differentiation.

Animals↗
PubMed1985

Gene expression in mice after high efficiency retroviral-mediated gene transfer.

A retroviral expression vector (N2) containing the selectable gene, neoR, has been used to determine the optimal conditions for infecting murine hematopoietic progenitor cells at high efficiency. After infected bone marrow cells were introduced into lethally irradiated mice, the presence, stability, and expression of the vector DNA sequences were analyzed either in individual spleen foci 10 days later or in the blood, bone marrow, and spleens of mice 4 months later. When bone marrow cells were cultured in medium containing virus with titers of more than 10(6) colony-forming units per milliliter in the presence of purified murine interleukin-3, more than 85 percent of the resulting foci contained vector DNA. This proviral vector DNA was intact. Efficient expression of the neoR gene was demonstrated in most of the DNA-positive foci examined. The spleens of reconstituted animals (over a long term) contained intact "vector DNA" and the blood and bone marrow expressed the neoR gene in some animals. Thus, a retroviral vector can be used to introduce intact exogenous DNA sequences into hematopoietic stem cells with high efficiency and with substantial expression.

Animals↗
PubMed1983

Murine embryonal carcinoma cells differentiate in vitro in response to retinol.

In an initial effort to determine whether circulating retinol might promote differentiation of embryonal carcinoma (EC) cells in tumor form, we have assessed the ability of retinol to stimulate differentiation of cultured EC cells. We found that retinol induces several murine EC cell lines to differentiate in vitro. Differentiated derivatives were distinguishable from parental EC cells by morphology, cell surface antigenic properties and levels of secretion of plasminogen activator. Retinol effects could be seen at concentrations as low as 8.7 X 10(-8) M (0.025 microgram/ml). Only two of eight EC lines tested failed to differentiate in response to retinol: PCC4-aza1R, which dies at retinol concentrations above 3.5 X 10(-7) M; and PCC4(RA)-1, a mutant line derived from PCC4-aza1R cells, which also fails to differentiate in response to retinoic acid.

Animals↗
PubMed1980

Formation of tetraploid mouse blastocysts following blastomere fusion with polyethylene glycol.

A rapid method to produce tetraploid embryos by blastomere fusion using polyethylene glycol (PEG) has been developed. Individual four-cell stage blastomeres were aggregated into pairs with phytohemagglutinin (PHA) and then treated with 45% (w/v) PEG (MW 1,000). 56.8% (804/1,416) of treated blastomere pairs fused. Tetraploid blastomeres were aggregated into quartets or doublets to restore cell number equivalent to that of whole or half diploid embryos, respectively. Best development was obtained with quartets, 72.9% (78/107) of which cavitated. 46.4% (13/28) of doublets and 31.7% (13/41) of singly cultured tetraploid blastomeres formed cavitated structures. Uniform tetraploidy was confirmed by fixing cavitated embryos and analyzing their metaphase plates. The fusion method described here replaces Sendai virus with a chemically defined fusogen and confirms earlier observations (Snow, '73; Graham, '71) that tetraploidy does not prohibit preimplantation development. This fusion method obviates some of the drawbacks of cytochalasin B-induced tetraploidy, namely the blocking of cleavage and the potential of diploid/tetraploid mosaicism.

Animals↗