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Biomedical subjects

R C Mulligan

Publications and source records attributed to R C Mulligan.

At least 91 records · Page 5Linked to original sources
PubMed1990

Temporary amelioration of hyperlipidemia in low density lipoprotein receptor-deficient rabbits transplanted with genetically modified hepatocytes.

Familial hypercholesterolemia is an inherited disease in humans that is associated with coronary artery disease and is caused by a deficiency of the receptor that mediates the internalization of low density lipoprotein (LDL). We have used an animal model for familial hypercholesterolemia, the Watanabe heritable hyperlipidemic (WHHL) rabbit, to design a therapeutic approach for this disease, which attempts to correct the hepatic defect in LDL receptor expression. Hepatocytes were harvested from WHHL rabbits, plated in primary cultures, and exposed to recombinant retroviruses capable of efficiently transferring a functional human LDL receptor gene. Genetically modified cells were harvested and infused into the portal vein of WHHL recipients, who were analyzed for metabolic consequences of human LDL receptor expression. Each animal exhibited a statistically significant decrease in total serum cholesterol 2-6 days after transplantation, with an eventual return to pretreatment levels. Proviral DNA sequences and virus-directed transcripts were detected in liver tissue 24 hr after transplantation. In situ hybridization demonstrated provirus expression in a small population of hepatocytes distributed in periportal sections of the liver. This study illustrates the potential of somatic gene therapy in ameliorating hyperlipidemia associated with familial hypercholesterolemia.

Animals↗
PubMed1989

Implantation of vascular grafts lined with genetically modified endothelial cells.

The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

Animals↗
PubMed1989

Repression of the IgH enhancer in teratocarcinoma cells associated with a novel octamer factor.

Embryonal carcinoma (EC) cell lines are models for early cells in mouse embryogenesis. A 300-base pair fragment of the heavy chain enhancer was inactive in F9 EC cells, unlike in other nonlymphoid cells where it has significant activity. Alterations of the octamer motif increased enhancer activity. Nuclear extracts from F9 cells contained an octamer binding protein (NF-A3) that was unique to EC cells; the amount of NF-A3 decreased upon differentiation. It is proposed that NF-A3 represses specific regulatory sequences that contain the octamer motif. Thus, the same DNA sequence mediates either negative or positive transcriptional effects, depending on the cell type.

Animals↗
PubMed1989

Restoration of growth potential in paraclones of human keratinocytes by a viral oncogene.

Human diploid keratinocytes may be divided into three clonal types with differing capacities for proliferation. The paraclone, which has the shortest life span, is limited to 15 divisions, after which all the cells undergo programmed terminal differentiation. By means of a retroviral vector, paraclones which have not completed their life span and which consist of not more than a few hundred cells can be transduced at a high frequency with DNA complementary to the 12S transcript of the adenovirus early region 1A gene. Transformation can be detected within a single cultivation by the formation of progressively growing colonies. The transformants appear to have an unlimited growth potential, and they form a disorganized epidermis when they are grafted as an epithelium onto athymic mice. These experiments clearly show that, in order to be transformed by a viral oncogene, the target cell need not be a stem cell.

Aged↗
PubMed1989

Antisense RNA complementary to 3' coding and noncoding sequences of creatine kinase is a potent inhibitor of translation in vivo.

Antisense RNA is a potentially powerful tool for creating dominant negative mutations, but one of the limitations of this strategy has been the relative inefficiency of antisense transcripts in blocking target gene expression. To identify more effective target sequences, helper-free retrovirus-mediated gene transfer was used to introduce antisense RNAs complementary to multiple functional regions of the human creatine kinase B (CK-B) mRNA into U937 cells. Antisense RNA complementary to the last third of the coding and all of the noncoding regio of this mRNA is highly effective; one or two antisense transcripts is sufficient to block the expression of one CK-B mRNA. In contrast, antisense RNA from which sequences complementary to the last 17 codons and all the 3' noncoding region have been deleted has no effect on CK-B expression. Neither antisense RNA alters the abundance of the target message, processing of the primary transcript, egress of the CK-B message from the nucleus, or the polysome profile of CK-B mRNA in sucrose gradients. These results point to a direct effect of the antisense transcript on translation and suggest that this effect may be explained at least in part by an inhibition of elongation or termination as a consequence of the duplex formed in the distal coding and/or 3' noncoding region.

Blotting, Northern↗
PubMed1989

Transfer of a mutant dihydrofolate reductase gene into pre- and postimplantation mouse embryos by a replication-competent retrovirus vector.

In order to explore the potential of retrovirus vectors for efficiently transferring foreign genes into mouse embryos, a replication-competent recombinant Moloney murine leukemia virus (Mo-MLV) vector carrying a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat was used to infect pre- and postimplantation embryos. When preimplantation mouse embryos were infected with the vector, as expected, the provirus integrated into the embryos and the germ line with the same efficiency as that observed with wild-type Mo-MLV, leading to inactivation of the recombinant virus. In contrast, when postimplantation mouse embryos were microinjected with virus-producing cells, between 90 to 100% of the surviving animals proved to be infected with the virus. The recombinant virus spread as efficiently as wild-type Mo-MLV in the infected embryos, resulting in up to three to five proviral copies per genome in heart, thymus, and brain tissues. Substantial expression of mutant DHFR*-coding viral message was found in all somatic tissues analyzed, the amounts correlating with the proviral copy number in the respective organ. These results suggest that replication-competent vectors are useful for efficient transfer and expression of foreign genes into tissues or whole animals when virus spread is needed.

Animals↗
PubMed1989

Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance.

A series of replication-competent Moloney murine leukemia virus vectors was constructed in which each vector contained a mutant dihydrofolate reductase (DHFR) cDNA insert in the U3 region of the viral long terminal repeat. Two of the resulting viruses, MLV (murine leukemia virus) DHFR*-5 and MLV DHFR*-7, were able to stably transfer methotrexate resistance to infected fibroblast cells upon multiple rounds of virus replication and in the absence of drug selection. Cell lines producing recombinant virus with high titers were established, which indicated that the insert did not grossly interfere with viral replication functions. These vectors should be useful for introducing and expressing foreign genes in vivo in tissues and whole animals in which virus spread is needed for efficient infection.

Animals↗
PubMed1988

Retroviral-mediated transfer and expression of human beta-globin genes in cultured murine and human erythroid cells.

We have cloned human beta-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 beta-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the beta-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human beta-globin gene transferred into mouse erythroleukemia cells in the same way. We have also introduced the normal human beta-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal beta-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, we suggest that the lack of expression of the endogenous beta-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for beta-globin gene transcription. Retroviral-mediated transfer of the human beta-globin gene may, however, uniquely influence expression of the gene in K562 cells.

Animals↗
PubMed1988

Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells.

Recombinant retroviral genomes encoding a chromosomal human beta-globin gene have been used to transduce murine haematopoietic stem cells in vitro. After permanent engraftment of lethally irradiated recipients with the transduced cells, the human beta-globin gene is expressed at significant levels only within the erythroid lineage. These results indicate that it is possible to obtain stable expression of exogenous chromosomal DNA sequences introduced into mature haematopoietic cells in vivo via stem cell infection, and that human disorders of haemoglobin production may be more feasible candidates for somatic cell gene therapy than previously suspected.

Animals↗
PubMed1988

Transfer of functional EGF receptors to an IL3-dependent cell line.

Epidermal growth factor (EGF) is a small protein that acts as a mitogen for various epidermal, epithelial, and fibroblastic cells that bear specific EGF receptors. The molecule that binds EGF is a 175-kD transmembrane protein, with an extracellular ligand binding domain and an intracellular domain that possesses tyrosine kinase activity, thought to be involved in the mitogenic signalling process. Here we have constructed a recombinant murine retrovirus that transduces a human cDNA encoding the 175-kD protein and used this retrovirus to infect BAF3, a murine, bone marrow-derived cell line, which is dependent on the haematopoietic factor interleukin-3 (IL3) for its growth in culture. The EGF receptors expressed in the infected cells exhibit two affinity states, as well as EGF-stimulated autophosphorylation. Furthermore, EGF can replace IL3 in supporting short-term proliferation of these cells. These data identify functional properties of the EGF receptor upon expression of the 175-kD EGF binding protein in a haemotopoietic cell that does not express endogenous receptors. They also suggest that gene transfer of growth factor receptors to heterologous cells may allow novel growth stimuli to be exploited.

Animals↗
PubMed1988

Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit.

Familial hypercholesterolemia is an inherited disease in humans that is caused by a defect in the receptor for low density lipoproteins (LDLR). The existence of an animal model for this disease, the Watanabe heritable hyperlipidemic (WHHL) rabbit, makes it an attractive candidate for developing new therapies that involve gene transfer into liver. As a first step toward the development of these therapies, we report the use of retrovirus-mediated gene transfer to correct the genetic defect in hepatocytes isolated from WHHL rabbits. A series of retroviral vectors that express the gene for human LDLR were constructed, each differing in the transcriptional elements used to drive LDLR expression. Helper-free amphotropic virus stocks representing each construct were then used to infect primary cultures of hepatocytes that were isolated from newborn WHHL rabbits. The efficiency of transduction, as measured by Southern analysis of integrated proviral sequences, ranged from 20% to 100%. Expression of human LDLR was analyzed by blot hybridization analysis of total cellular RNA and by biochemical and in situ analyses of transduced cultures for receptor function. The vector in which the expression of LDLR was driven by the viral long terminal repeat sequence produced the greatest quantity of LDLR RNA and protein in WHHL hepatocytes; LDLR activity approached normal levels in these cultures.

Animals↗
PubMed1988

Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.

We have constructed a set of packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. To eliminate the problems of transfer of packaging functions and helper virus formation encountered with the previously available packaging systems, two mutant Moloney murine leukemia virus-derived proviral genomes carrying complementary mutations in the gag-pol or env regions were sequentially introduced into NIH 3T3 cells by cotransformation. Both genomes contained a deletion of the psi sequence necessary for the efficient encapsidation of retroviral genomes into virus particles and additional alterations at the 3' end of the provirus. We show that the resulting packaging cell lines psi CRIP and psi CRE can be used to isolate clones that stably produce high titers (10(6) colony-forming units/ml) of recombinant retroviruses with amphotropic and ecotropic host ranges, respectively. More importantly, we demonstrate that viral producers derived from the packaging cell lines do not transfer the packaging functions, or yield helper virus, even under conditions where existing packaging cell lines can be shown to yield transfer of packaging functions and/or helper virus. These properties of the psi CRIP and psi CRE packaging lines make them particularly valuable reagents for in vivo gene transfer studies aimed at cell lineage analysis and the development of human gene replacement therapies.

Animals↗
PubMed1988

Two dominant-acting selectable markers for gene transfer studies in mammalian cells.

We report the development of two dominant-acting genetic markers useful for monitoring gene transfer in mammalian cells that are based on prokaryotic genes encoding key steps in the synthesis of the essential amino acids, tryptophan and histidine. Under appropriate conditions, expression of these genes obviates the nutritional requirements for their respective amino acid products. Expression of the trpB gene of Escherichia coli, which encodes the beta subunit of tryptophan synthase (EC 4.2.1.20), allows mammalian cell survival and multiplication in medium containing indole in place of tryptophan. The hisD gene of Salmonella typhimurium encodes histidinol dehydrogenase (EC 1.1.1.23), which catalyzes the two-step NAD+-dependent oxidation of L-histidinol to L-histidine. In medium lacking histidine and containing histidinol, only mammalian cells expressing the hisD product survive. The selection is a double one in that the provided precursor histidinol is itself toxic to animal cells through its inhibition of histidyl-tRNA synthetase; thus, the dehydrogenase both removes an inhibitor and forms a required end product. Alternatively, the his selection may be carried out under conditions in which the dehydrogenase serves mainly to detoxify histidinol. For either the trp or his selections the substitute nutrient (indole or histidinol) is readily available, inexpensive, stable, permeable to cells, and convertible to the end product in a step controlled by a single gene. Vectors based upon murine retrovirus and papovavirus backbones have been successfully employed for both genes, allowing selection in a range of cell types, including 3T3, CV-1, and HeLa. These dominant selective schemes should provide generally useful and inexpensive alternatives to others currently in use, such as the gpt, neo, hygro, dhfr, and tk selections.

Alcohol Oxidoreductases↗
PubMed1988

Retroviral transfer of a murine cDNA for multidrug resistance confers pleiotropic drug resistance to cells without prior drug selection.

We have constructed a retrovirus expression vector that carries the murine mdr cDNA transcribed under the control of the human H4 histone promoter to examine the feasibility of efficiently transferring a multidrug resistance phenotype to cells without requiring drug selection. This approach will facilitate the transfer of mdr cDNA to hematopoietic progenitor cells for the study of multidrug resistance in vivo. The retrovirus vector pHmdr has been used for transmission and expression of the mdr cDNA in initially drug-sensitive NIH 3T3 fibroblasts. Selection of pHmdr infectants in the cytotoxic agents colchicine or doxorubicin gave rise to highly multidrug-resistant colonies containing a single gene copy of the vector. Moreover, in the analysis of 12 cloned unselected NIH 3T3 cell infectants, a multidrug resistance phenotype was conferred by as few as two copies of the pHmdr vector. Overexpression of the mdr cDNA in drug-selected and unselected pHmdr infectants was directly related to cell survival in three cytotoxic agents tested. These results hold significant implications for the study of multidrug resistance in vivo.

Animals↗
PubMed1988

Retrovirus-mediated transduction of adult hepatocytes.

Retrovirus-mediated gene transfer was used to develop a method for introducing genes into primary cultures of adult rat hepatocytes. Subconfluent monolayers of hepatocytes, cultured in hormonally defined media on different matrix substrata, were infected with helper-free stocks of a replication-defective retrovirus that constitutively expresses high levels of beta-galactosidase. Retrovirus-mediated transduction was measured by two methods: (i) an in situ cytochemical stain that specifically detects the expression of viral expressed beta-galactosidase, and (ii) Southern blot analysis, which measures the relative copy number of integrated provirus. Maximal transduction efficiency of approximately equal to 25% was achieved when the cells were infected after 3 days in culture; matrix had little effect on transduction efficiency. Enzyme cytochemical (catalase and glucose 6-phosphatase) and peroxidase immunocytochemical (asialoglycoprotein and UDP-glucuronosyltransferase) analyses of the cultures indicated that greater than 95% of cells were hepatocytes. The demonstration of hepatocyte-specific organelles in cells expressing the viral-directed beta-galactosidase provided unambiguous evidence for the transduction of hepatocytes. These methods should be useful in the development of liver-directed somatic gene therapy and in the study of liver-specific gene regulation.

Animals↗
PubMed1988

Use of retroviral vectors for mapping of splice sites in cottontail rabbit papillomavirus.

Cottontail rabbit papillomavirus (CRPV) genomic sequences coding for virus early functions were introduced into a retroviral vector in order to produce cDNAs of the viral early region. Two constructs differing in the length of control sequences preceding the E6 open reading frame were transfected into Psi-2 cells and the released retroviral stock was used to infect NIH3T3 cells. The proviral sequences were rescued from antibiotic G418-resistant virus-infected cells after fusion with Cos cells, amplified as plasmids in Escherichia coli and analysed. Nucleotide sequencing showed that the splicing signals used in the construct containing only the early coding region are the same as in CRPV-expressing tumours, linking the beginning of E1 to the middle of E2. On the other hand, in a construct including most of the long control region a splice donor site located in the 5' end of this region, at position 7810, was very efficiently used, totally excluding the use of the donor site at position 1371. None of the constructs containing CRPV sequences transcribed from Moloney murine leukaemia virus promoter was able to transform mouse fibroblasts after DNA transfection.

Base Sequence↗