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Biomedical subjects

D Steffen

Publications and source records attributed to D Steffen.

9 recordsLinked to original sources
PubMed1992

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

Animals↗
PubMed1987

The sequence of a sea urchin muscle actin gene suggests a gene conversion with a cytoskeletal actin gene.

We report the nucleotide sequence of the single muscle actin gene of the sea urchin Strongylocentrotus purpuratus. Comparison of the protein-coding sequence of this muscle actin gene (pSpG28) with that of two linked sea urchin cytoskeletal actin genes (pSpG17 and CyIIa) reveals a region of exceptional sequence conservation from codon 61 through codon 120. Furthermore, when silent nucleotide changes are compared, the conservation of this region is still evident (7.9% silent site differences in the conserved region vs 43.3% silent site differences in the rest of the gene when pSpG28 and CyIIa are compared), indicating that the conservation is not due to particularly stringent selection on the portion of the protein encoded by this region of the genes. These observations suggest that a gene conversion has occurred between the muscle actin gene and a cytoskeletal actin gene recently in the evolution of the sea urchin genome. Gene conversion between nonallelic actin genes may thus play a role in maintaining the homogeneity of this highly conserved gene family.

Actins↗
PubMed1986

Multiple chromosomal rearrangements in a spontaneously arising t(6;7) rat immunocytoma juxtapose c-myc and immunoglobulin heavy chain sequences.

Spontaneously arising immunocytomas in Lou/Wsl rats contain a consistent translocation between chromosomes 6 and 7. The c-myc gene has been localized to chromosome 7 and has been shown to be rearranged in the majority of the rat immunocytomas. We now report the cloning of the rearranged 11-kilobase EcoRI c-myc fragment from the IgE-secreting IR75 tumor. Sequence analysis revealed that the cytogenetically visible t(6;7) translocation must have involved several events in this tumor. One event has led to the juxtaposition of c-myc and the switch mu region, in a head-to-head orientation. The breakpoint is approximately 850 base pairs upstream from the proximal c-myc promoter on chromosome 7. This area is distinct from the more common mouse plasmacytoma- and Burkitt lymphoma-associated translocation breakpoints and also differs from the known murine retroviral insertion sites. A second rearrangement has led to the transposition of sequences upstream from the switch gamma 1 region to the c-myc-distant end of the switch mu region, tail-to-tail. This requires at least two events, including one inversion. In addition to showing that identical loci (c-myc, immunoglobulin) are juxtaposed via chromosomal translocations in three different tumors (Burkitt lymphoma, mouse plasmacytoma, and rat immunocytoma) in different species (human, mouse, and rat), the multiple rearrangements in IR75 and some other tumors emphasize the selective value of c-myc activation by an immunoglobulin locus in the tumorigenic process.

Animals↗
PubMed1979

Identification of DNA fragments carrying ecotropic proviruses of AKR mice.

The proviruses of the N-tropic, ecotropic virus (AKV) of AKR mice (Akv-1, Akv-2) have been studied by the Southern gel--filter transfer technique. These proviruses can be detected by cleavage of cell DNA by BamHI endonuclease, which yields characteristic subgenomic DNA fragments upon cleavage of this type of provirus. Proviruses integrated into different sites in the mouse genome can be resolved with EcoRI endonuclease, which does not cleave the AKV proviruses. Use of congenic and backcrossed mice and a radioactive DNA probe enriched for AKV sequences has allowed identification of the EcoRI fragments carrying the proviruses of the genetically defined Akv-1 and Akv-2 loci. Novel proviruses introduced by superinfection of cultured AKR cells with AKV and present in leukemic cells from AKR mice have also been identified. Comparison of substrains of AKR mice indicates some heterogeneity in their spectra of proviruses.

AKR murine leukemia virus↗
PubMed1978

The integrated genome of murine leukemia virus.

The Southern gel filter transfer technique has been used to characterize the integrated genome of Moloney murine leukemia virus (M-MuLV) and the genomes of the endogenous viruses of the mouse. Study of 10 clones of rat cell independently infected by M-MuLV indicates a minimum of 15 integration sites into which the M-MuLV provirus can be inserted. No common integration site is observed among these clones. Clones productively infected by M-MuLV acquire multiple proviruses, whereas infected cells unable to produce virus contain only one M-MuLV provirus. Once established, the integrated genomes are stable for at least two years after initial infection. The use of M-MuLV probe allows detection of a spectrum of Eco RI-cleaved mouse DNA fragments containing endogenous MuLV genomes. DNAs of different inbred laboratory mouse strains yield similar patterns of provirus with each strain showing minor characteristic differences. In some instances, mouse cells infected by M-MuLV reveal additional proviruses beyond those seen in the uninfected cell. DNAs from three different M-MuLV-induced thymomas indicate, as in rat cells, multiple possible integration sites.

Animals↗
PubMed1977

Overproducing araC protein with lambda-arabinose transducing phage.

Escherichia coli infected with bacteriophage lambda-arabinose transducing phage were tested as sources of araC protein. Infection of cells with such phage produces an intracellular concentration of araC protein up to 100 times that present in wild-type E. coli, apparently resulting from fusion of the araC gene to bacteriophage lambda promoters. Lysates from these phage-infected cells may be fractionated to yield another 100-fold enrichment in araC activity so that the total enrichment is 10,000-fold. A nonsense mutation in araC provided proof of the identification on gel electrophoresis of a band in the purified material. Biologically active araC protein is a dimer with 28,000 M.W. subunits. The araC gene in these phage replaces the int-xis genes but is oriented in the opposite direction. Nonetheless, it appears to be transcribed in this position by the phage promoter pr via transcription the long way around. Furthermore, because araC gene is in this position, we were able to isolate phage on which the araC gene was under phage late gene control by deletion of the late gene transcription stop signals in the b2 region.

Arabinose↗