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Biomedical subjects

R G Hawley

Publications and source records attributed to R G Hawley.

102 records · Page 6Linked to original sources
PubMed1984

Biochemical genetics of the mouse IgM system.

Using a mouse hybridoma system, we have developed methods of isolating a variety of mutant cell lines in which immunoglobulin function or synthesis is defective. The analysis of mutants defective in kappa chain synthesis has defined a class of murine transposons. The deletion mutants produce immunoglobulin M (IgM) bearing mu heavy chain fragments and provide information on the requirements of IgM assembly and mu gene expression. We also describe a transfer system for the mu and kappa genes which will be useful in analyzing the structural basis of IgM function.

Animals↗
PubMed1983

Functional immunoglobulin M production after transfection of cloned immunoglobulin heavy and light chain genes into lymphoid cells.

The rearranged immunoglobulin heavy (mu) and light (kappa) chain genes cloned from the Sp6 hybridoma cell line producing immunoglobulin M specific for the hapten 2,4, 6-trinitrophenyl were inserted into the transfer vector pSV2-neo and introduced into various plasmacytoma and hybridoma cell lines. The transfer of the mu and kappa genes resulted in the production of pentameric, hapten-specific, functional IgM.

Animals↗
PubMed1982

Mutant immunoglobulin genes have repetitive DNA elements inserted into their intervening sequences.

The kappa light chain genes from two mutant hybridoma cell lines defective in kappa light chain synthesis were isolated and compared to the wild-type kappa light chain gene. In each case, the mutant kappa light chain genes were found to contain repetitive DNA elements in their intervening sequences that were not present in the intervening sequences of the wild-type kappa light chain gene. These elements were found to be related to the genes of intracisternal A particles. These results suggest that the decreased production of kappa light chain in the mutant cell lines is due to the presence of the intracisternal A particle-related genes.

Animals↗
PubMed1981

Molecular cloning of an immunoglobulin kappa constant gene from NZB mouse.

An EcoRI fragment carrying the immunoglobulin C kappa gene and multiple J gene segments from the DNA of the NZB strain of mouse was cloned into lambda Ch 4A DNA. Subsequent characterization of the clone by heteroduplex analysis, restriction-enzyme mapping and DNA sequencing demonstrated that the organization of the J gene segments and the C kappa gene of NZB mouse was similar, if not identical, to that of DNA from the Balb/c strain of mouse. Since the amino acid sequence of the light chain of a plasmacytoma of NZB mouse shows a J region sequence different from that of Balb/c mouse, our results indicate that the new J sequence arose by somatic mutation.

Animals↗
PubMedDate not supplied

Retroviral transduction of human CD34+ cells on fibronectin fragment CH-296 is inhibited by high concentrations of vector containing medium.

BACKGROUND: The objective of the present study was to optimize conditions for retroviral transduction of human cord blood (CB) CD34+ cells and to reveal mechanisms which interfere with efficient gene transfer. METHODS: An MSCV based retroviral vector with the gene for enhanced green fluorescent protein (MGIN) produced by GP+envAM12 (amphotropic envelope), PG13 (gibbon ape leukemia virus envelope) or 293GPG (vesicular stomatitis virus envelope) cell lines was used to transduce cord blood CD34+ cells on Retronectin (fibronectin fragment CH-296) in three different ways: either in vector containing medium (VCM), in fresh medium on Retronectin pre-loaded with vector or in VCM on Retronectin pre-loaded with vector. RESULTS: Paradoxically, the transduction efficiency obtained with pre-load of vector onto Retronectin alone was higher than pre-load plus VCM for PG13-MGIN (67.9 +/- 6.0% vs 24.9 +/- 8.0%) and AM12-MGIN (47.5 +/- 5.8% vs 38.7 +/- 2.2%). Further experiments showed that transduction on Retronectin pre-loaded with PG13-MGIN or AM12-MGIN was inhibited by the presence of the same VCM at high concentrations, but not by the presence of a VCM with a different receptor specificity. If no pre-load of vector was performed, the highest transduction efficiencies were seen when VCMs were diluted 1:10 (MOIs of 3). The inhibitory effect of high titer PG13-MGIN VCM was confirmed in more primitive CD34+CD38low cells and in NOD/SCID repopulating cells, and was also seen in experiments with bone marrow CD34+ cells. CONCLUSIONS: Retroviral transduction of CB CD34+ cells on Retronectin is inhibited by high titer PG13 and GP+envAM12 vector containing medium. Efficient gene transfer to human hematopoietic cells can be obtained by preload alone of the vector onto Retronectin. These findings are of importance for the design of transduction protocols for repopulating hematopoietic cells.

Antigens, CD34↗
PubMedDate not supplied

Transfer of a cloned immunoglobulin light-chain gene to mutant hybridoma cells restores specific antibody production.

The expression of immunoglobulin (Ig) genes is regulated at several levels. For example, although kappa-chain production requires a DNA rearrangement that juxtaposes variable and joining segments, this rearrangement is not sufficient for kappa-chain gene expression; that is, some cell types do not permit immunoglobulin production. The mechanisms responsible for the regulation of the expression of rearranged immunoglobulin genes are poorly understood. The technique of modifying cloned genes in vitro and transferring the modified genes to cells in culture provides a tool for identifying the structural features required for gene expression. To analyse immunoglobulin genes in this manner, however, it is first necessary to use, as recipients, cells that normally permit immunoglobulin production. We report here that a cloned kappa-chain gene is expressed in immunoglobulin-producing hybridoma cells. Furthermore, the product of the transferred kappa-chain gene is capable of restoring specific antibody production to the transformed cells.

Animals↗
PubMedDate not supplied

Retroviral-mediated transfer of genes encoding interleukin-2 and interleukin-12 into fibroblasts increases host antitumor responsiveness.

The transfer of genes encoding cytokines into tumor cells has emerged as a new strategy to increase in vivo host reactivity to a variety of tumors. Because gene transfer into tumor cells cannot be easily applied in the clinical setting, we have developed an experimental model of gene transfer into fibroblasts and examined the capacity of these engineered cells to elicit an antitumor immune response. Interleukin-12 (IL-12) is a heterodimeric cytokine with pleiotropic activities presenting strong antitumor and antimetastatic effects in murine models. A bicistronic retroviral vector was constructed that contained the cDNAs encoding both chains (p40 and p35) of murine IL-12 separated by an internal ribosomal entry site sequence. Syngeneic cutaneous fibroblasts obtained from newborn mice and transduced to secrete either IL-12 or IL-2 were injected subcutaneously with B16F0 or B16F1 melanoma cells. The time of tumor occurrence and overall survival of mice were significantly prolonged when B16F1 cells were coinjected with cytokine-producing fibroblasts compared with B16F1 alone or B16F1 together with unmanipulated fibroblasts. Systemic effects were seen in the mice injected with either IL-2- or IL-12-secreting fibroblasts, with the highest proliferation capability and interferon-gamma production observed in vitro from splenocytes from recipients of IL-2-secreting fibroblasts. Injection of IL-2-secreting fibroblasts or coinjection of IL-2- and IL-12-producing fibroblasts resulted in a significant increase of survival in the B16F0 model; in some cases, complete disease eradication was observed. These results suggest that cutaneous fibroblasts represent a target of choice for gene transfer and would be useful in the treatment of minimal residual disease in humans.

Animals↗
PubMedDate not supplied

Transmissibility of murine stem cell virus-based retroviral vectors carrying both interleukin-12 cDNAs and a third gene: implications for immune gene therapy.

The combination of immunotherapy with conventional treatments such as radio- and chemotherapy may be necessary to eradicate minimal residual disease. Interleukin 12 (IL-12) is a heterodimeric cytokine composed of two subunits, p40 and p35. Coordinate expression of the IL-12 p40 and p35 genes in several solid tumor models has been found to induce strong and specific antitumor immune responses. In the interest of obtaining high level IL-12 expression in leukemia/lymphoma cells for use as vaccines in cancer immunotherapy, we evaluated three IL-12 retroviral vector designs based on the murine stem cell virus (MSCV) vector which efficiently transduces functional genes into normal hematopoietic cells. MSCVpac-mlL-12 and MIPV-mIL-12 contain an encephalomyocarditis virus internal ribosome entry site for internal translation of bicistronic mRNA transcripts, while MDCVpac-mIL-12 carries an expression cassette in the U3 region of the 3' long terminal repeat. We found that the MSCVpac-mIL-12 vector directed robust expression of both p40 and p35 genes in several murine tumor cell lines of hematopoietic origin, including a T-cell lymphoma, a B-cell lymphoma, and a plasmacytoma/myeloma. In contrast, genomic instability or promoter interference hampered p40 gene expression in cells transduced with the MIPV-mIL-12 and MDCVpac-mIL-12 vectors, respectively. These findings provide the basis for the design of IL-12 retroviral vectors for the treatment of hematologic malignancies in humans.

Acetyltransferases↗