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Biomedical subjects

E F Wagner

Publications and source records attributed to E F Wagner.

At least 145 records · Page 8Linked to original sources
PubMed1988

Expression of human alpha 1-acid glycoprotein genes in cultured cells and in transgenic mice.

The human genome contains three alpha 1-glycoprotein genes (AGP-A, AGP-B, and AGP-B') encoding for slightly different forms of the protein. The major component of human alpha 1-acid glycoprotein found in plasma is coded by AGP-A, which is expressed in liver and in hepatoma cell lines and is induced by inflammatory stimuli. We have studied the regulation of the cloned AGP-A gene by transfection into cell lines of hepatic and nonhepatic origin. Unlike any other liver-specific gene investigated so far, every AGP construct tested was expressed with comparable efficiency in hepatoma and HeLa cells. In contrast, identical constructs in transgenic mice are expressed in a tissue-specific manner and are regulated by acute-phase stimuli. Transgenic mice carrying the cluster of three AGP genes secrete the human protein in the serum, and the corresponding mRNA is mainly derived from the AGP-A gene. The mRNA is liver specific, and its concentration increases several fold following experimentally induced inflammation. Additional transgenic lines carrying only the AGP-A gene showed that sufficient information for tissue-specific and regulated expression is contained within a 6.6-kb segment comprising the whole coding region plus 1.2-kb 5'-flanking and 2-kb 3'-flanking DNA.

Animals↗
PubMed1988

The effects of v-src expression on the differentiation of embryonal carcinoma cells.

We have used replication-defective selectable retroviruses to express inducibly or constitutively the v-src gene in the embryonal carcinoma (EC) cell lines F9, PC13 and P19. High v-src expression in F9 and PC13 cells does not induce or disrupt their differentiation. In contrast, P19 cells expressing high levels of v-src have a 'differentiated' morphology and are unable to respond to signals that normally induce differentiation along the neural or muscle pathways. Regulation of v-src transcription from the inducible human metallothionein (MT) promoter has shown that v-src expression above a threshold level induces differentiation of these cells, as defined by loss of the stem cell marker ECMA-7. These results suggest that v-src expression may be compatible with differentiation events occurring early in embryogenesis, represented by F9 and PC13 cell differentiation, but might disrupt the differentiation of cell types present at later developmental stages.

Animals↗
PubMed1987

Inducible and tissue-specific expression of human C-reactive protein in transgenic mice.

C-reactive protein (CRP) is a major acute phase reactant in man but not in mouse. It is synthesized in abundant quantities by human hepatocytes during the course of several diseases, mainly acute inflammations. To investigate the regulation of CRP expression, the human CRP gene was introduced into fertilized eggs by microinjection and transgenic mouse lines were derived. The CRP gene is exclusively transcribed in the liver and expression is strictly dependent on experimental inflammation. The kinetics of induction both for RNA and protein synthesis is very fast; RNA is first detectable after 2 h in the liver, the protein after 6 h in the serum. Human CRP levels in the sera of transgenic mice are comparable to those observed in human diseases. Nuclear run-on experiments indicate that regulation is primarily at the transcriptional level.

Animals↗
PubMed1987

The human alpha-1-antitrypsin gene is efficiently expressed from two tissue-specific promotors in transgenic mice.

Alpha 1-antitrypsin (alpha 1AT) present in large amounts in human serum and synthesized predominantly in hepatocytes, is the most abundant protease inhibitor and alpha 1AT mutant proteins are associated with hereditary disorders. To investigate the regulation of the normal human alpha 1AT gene, we have microinjected fertilized mouse eggs with a 17.5 kb DNA fragment containing the entire gene with 7 kb 5' and 0.3 kb 3' flanking sequences. We show that this DNA fragment contains all the information for efficient, accurate and tissue-specific expression. High serum concentration of the human protein was found in three independent transgenic mouse lines. The human alpha 1AT RNA is transcribed efficiently in liver, kidney and macrophages and we demonstrate that two different promoters are used for the expression in liver and macrophages of the transgenic mice.

Animals↗
PubMed1987

Expression of retroviral vectors in transgenic mice obtained by embryo infection.

Pre-implantation embryos were infected with the retroviral vector MMCV-neo, which carries the neomycin resistance (neo) gene and the v-myc gene. Three transgenic substrains (M-TKneo 1-3) were derived which stably transmit a single intact copy of the vector. In all of the substrains, expression of the neo gene from the internal thymidine kinase (TK) promoter was detected, with two of the substrains expressing the gene in all tissues analysed. In the third substrain, the vector had integrated on the X chromosome and neo expression varied between different tissues. A second series of transgenic mice were obtained with the retroviral vector SAX, in which the human adenosine deaminase cDNA (ADA) is under the control of an internal SV40 promoter. Four substrains (M-SAX 1-4) were analysed; however, no expression of the ADA cDNA was detected. In all mice, no expression was found of the genes under the control of the viral 5' long terminal repeats (LTRs). In the M-TKneo substrains the vector was hypomethylated irrespective of its expression whereas in the M-SAX mice the vector was hypermethylated. These results demonstrate for the first time that the TK promoter can apparently express a gene in all tissues of adult mice and that retroviral vectors with internal promoters may provide an alternative to DNA injection for the efficient expression of genes in transgenic mice.

Animals↗
PubMed1986

Introduction of a selectable gene into murine T-lymphoblasts by a retroviral vector.

A recombinant retroviral vector with an inserted bacterial neomycin resistance (neo) gene was used to transfer in vitro neomycin resistance (neoR) into murine cytotoxic lymphocyte precursors (CLP). The infection protocol involved co-cultivation of mitogen-activated splenic T-blasts with irradiated cells that produced either the recombinant retrovirus plus a helper virus, or exclusively the recombinant retrovirus. Infected T-blasts were subsequently cultured under limiting dilution (LD) conditions that supported clonal in vitro development of a large fraction of murine CLP. In infected T-blast populations, frequency estimates were obtained for CLP that developed into functional cytotoxic T-lymphocyte (CTL) populations under G418-selected or non-selected conditions; from these frequency estimates, an efficiency of transduction of the neoR phenotype into murine CLP of 2-8% was calculated. Some conditions were defined that influenced transduction efficiency, i.e., the density of the infecting monolayer cells; the presence of interleukin 2-containing conditioned medium and mitogenic lectins during the co-culture period; a delayed onset of G418 selection after infection. It was demonstrated that the neoR phenotype of functional CTL populations derived from infected CLP resulted from expressed recombinant retrovirus.

Animals↗
PubMed1986

Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.

A retrovirus-derived vector called self-inactivating (SIN) vector was designed for the transduction of whole genes into mammalian cells. SIN vectors contain a deletion of 299 base pairs in the 3' long terminal repeat (LTR), which includes sequences encoding the enhancer and promoter functions. When viruses derived from such vectors were used to infect NIH 3T3 cells, the deletion was transferred to the 5' LTR, resulting in the transcriptional inactivation of the provirus in the infected cell. Introduction of a hybrid gene (human metallothionein-promoted c-fos) into cells via a SIN vector was not associated with rearrangements and led to the formation of an authentic mRNA transcript, which in some cases was induced by cadmium. SIN vectors should be particularly useful in gene transfer experiments designed to study the regulated expression of genes in mammalian cells. Absence of enhancer and promoter sequences in both LTRs of the integrated provirus should also minimize the possibility of activating cellular oncogenes and may provide a safer alternative to be used in human gene therapy.

Chromosome Deletion↗
PubMed1985

Expression of foreign genes from retroviral vectors in mouse teratocarcinoma chimaeras.

The formation of chimaeric mice from embryonic stem cells (EC and ES) carrying and exposing foreign genes is described. Two retroviral vectors were used to stably introduce genes, by virus infection and selection into EC and ES cells. In the first series EC cell clones were isolated that contained a single intact copy of the vector, which expressed the neomycin resistance gene (neo) from the 5' long terminal repeat (LTR). Following the formation of chimaeras with one of the clones, expression of the foreign gene was found in all chimaeric tissues examined. In the second series, a vector was used which contained the OK10 v-myc oncogene under the control of the 5' LTR as well as the neo gene expressed from an internal thymidine kinase (TK) promoter. ES cell clones were again isolated and used to form chimaeras. Although expression of the TK neo transcripts was maintained in the chimaeric tissues analysed, no expression of the v-myc transcripts from the 5' LTR was detected. These results show that genes can be introduced into mice, using clones of embryonic stem cells selected in vitro and that the expression of the genes can be maintained probably throughout development and in all tissues. The advantages this approach offers compared with DNA injection into eggs and the use of retroviruses as vectors for introducing genes into mice are discussed.

Animals↗
PubMed1985

Transfer of genes into embryonal carcinoma cells by retrovirus infection: efficient expression from an internal promoter.

The expression of retroviral genomes after infection of embryonal carcinoma (EC) cells is usually restricted. We have constructed a retroviral vector which has the potential to express efficiently in EC cells an inserted sequence, the selectable neomycin (neo) gene, from an internal thymidine kinase promoter. A second retrovirus vector contained in addition a v-myc oncogene, which in NIH 3T3 cells could be expressed from the viral long terminal repeat (LTR) through a subgenomic mRNA. 10-100% of the EC cells infected with recombinant virus gave rise to neo-resistant colonies following drug selection. The neo gene was expressed in both the non-permissive EC and the permissive NIH 3T3 cells at similar levels, and transcription of neo-specific RNA was initiated at the proper site in the thymidine kinase promoter. In contrast, v-myc mRNA was produced only in the NIH 3T3 cells and not in EC cells, even after induction of differentiation. Thus, genes inserted into retrovirus constructs can easily be introduced into EC cells by virus infection, even without drug selection, and they can be efficiently expressed when transcribed from promoters other than the viral LTRs.

Animals↗
PubMed1985

Analysis of the differentiation-promoting potential of inducible c-fos genes introduced into embryonal carcinoma cells.

To investigate the differentiation-promoting potential of c-fos in embryonal carcinoma cells (EC cells) we have designed various human metallothionein promoter-mouse-c-fos gene constructs containing also the selectable SV40 promoter-driven neo gene. Upon transfection into F9 EC cells and selection for neo resistance, the following results were obtained. (i) With each of the constructs, colonies of morphologically altered and differentiated (i.e., TROMA-1 and TROMA-3 expressing) cells were identified. (ii) Expression of c-fos was required to affect the differentiation state of F9 cells to a significant extent, but a low level was sufficient; no enhancement of differentiation was noticeable even after 100-fold induction of c-fos expression by cadmium. (iii) F9 cell clones were isolated which, in spite of very high levels of exogenous c-fos expression, had stem cell morphology. These cells, however, continuously generated morphologically altered and differentiated cells upon subculturing. (iv) In other EC cell lines, which resemble stem cells more closely than the 'partially differentiated' F9 cells, c-fos expression showed either a less pronounced (P19 cells) or no differentiation-promoting effect at all (PC13 cells). Our results suggest that the c-fos gene product acts in concert with other, probably 'spontaneously' occurring events to promote differentiation of certain EC cell lines.

Animals↗
PubMed1985

Clustered homeo boxes are differentially expressed during murine development.

A murine homeo box (m6-12) known to be expressed during differentiation of embryonal carcinoma cells lies within a cluster of homeo boxes located in 30 kilobase pairs of genomic DNA. We have established the organization of the boxes within this complex, as well as the nucleotide sequence of a second box, m5-4. Similar to the m6-12 box, expression of novel m5-4 transcripts is induced upon differentiation of embryonal carcinoma cells. Transcripts of genes containing m6-12 were found in embryonic tissue during almost all stages of prenatal development studied, whereas expression of m5-4 was detected only in 12 day embryonic tissue. Finally, we have described the differential expression of these homeo-box-containing regions in various adult tissues.

Amino Acid Sequence↗
PubMed1984

Gene transfer into the germ line of mice and into embryonal carcinoma stem cells.

Recombinant genes can be introduced into the germ line of mice by microinjection into the fertilized egg or via embryonal carcinoma stem cells. A regulated, cell-type specific expression of the newly introduced gene following microinjection is usually not found. However, in one series of experiments the insertion of human growth hormone genes in the germ line led to prenatal recessive lethal mutations. Alternatively, embryonal carcinoma stem cells can be used to introduce and express genes in mice, for example after infection with selectable retroviral vectors. In addition, these cells are useful for differentiation studies in vitro following DNA-mediated gene transfer.

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↗