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H Westphal

Publications and source records attributed to H Westphal.

At least 127 records · Page 7Linked to original sources
PubMed1990

Synthesis of alpha-crystallin by a cell line derived from the lens of a transgenic animal.

Cultured cells derived from transgenic animals have not generally been utilized to investigate questions in cell biology; however, to study the properties of proteins from the lens of the eye, a cell line which synthesizes alpha-crystallin was established from a transgenic mouse. All the alpha-crystallins, alpha A, alpha B, and alpha Insert, accumulate in the cell line. The alpha-crystallin, 1.6% of the cellular protein, is found in large molecular weight aggregates similar to the aggregates found in the normal mouse lens. The alpha-crystallins in the lens cells correspond exactly to both the unmodified and the phosphorylated alpha-crystallins found in the lens of the mouse, suggesting that some post-translational modification of the mouse alpha-crystallin may be important to the structure of this protein.

Animals↗
PubMed1990

Differentiation versus proliferation of transgenic mouse lens cells expressing polyoma large T antigen: evidence for regulation by an endogenous growth factor.

Expression of the immortalizing oncogene polyoma large T antigen (PyLT) in the lens of transgenic mice impairs fiber cell differentiation but does not induce hyperplasia or tumor development. To examine this phenotype further, we studied the expression of the lens-specific crystallin genes in normal mice and mice of the transgenic alpha PyLT1 lineage. Immunochemical analyses showed that the spatial pattern of expression of alpha, beta, and gamma crystallins was abnormal in lenses from alpha PyLT1 mice, and that abnormally differentiated lens cells expressed beta and gamma crystallins. We also found that the levels of expression of the crystallin proteins in the transgenic mice were reduced compared to those in normal mice. In vitro, epithelial lens cells from alpha PyLT1 mice expressed not only PyLT and alpha crystallins, but also beta and gamma crystallins, which occur specifically in differentiated cells. Yet, despite their nonproliferative nature in vivo and their expression of differentiated lens cell markers, lens cells from alpha PyLT1 mice proliferated indefinitely in vitro. The growth of the alpha PyLT1 lens cells in culture was inhibited by treatment with exogenous basic fibroblast growth factor, which is thought to be involved in growth and differentiation of lens cells in vivo. These results suggest that factors in the microenvironment of the eye may be important in the process of tumor formation in vivo.

Animals↗
PubMed1989

Cell-specific induction of mouse Cyp1a1 mRNA during development.

The dioxin-inducible cytochrome P(1)450 (Cyp1a1 gene) and P(3)450 (Cyp1a2 gene) enzymes have been implicated in the metabolism of numerous polycyclic hydrocarbons and arylamines, respectively. The prototypic inducer 3-methylcholanthrene was given to the pregnant mouse, and the intrauterine response was examined with the use of in situ hybridization. During the early postimplantation stage, inducible Cyp1a1 mRNA is detected in specific cell types in the extraembryonic tissues only. This selective expression along with the lack of detectable constitutive Cyp1a1 and constitutive or inducible Cyp1a2 gene transcripts between 5.3 and 14.5 days of gestation suggest that (i) these two genes appear to play no endogenous role during differentiation and (ii) the metabolic activity of the inducible Cyp1a1 enzyme may be important to the embryo and fetus from the standpoint of protection against toxic foodstuff and other environmental chemicals.

Animals↗
PubMed1989

The human immunodeficiency virus long terminal repeat is preferentially expressed in Langerhans cells in transgenic mice.

Four lines of transgenic mice containing the HIV LTR linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT) were constructed. In each line, a characteristic tissue pattern of CAT expression was observed with detectable levels present in the eye, heart, spleen, thymus, and tail. Low levels of CAT were present in circulating lymphocytes, but CAT activity in these cells could be augmented following treatment with the mitogen phytohemagglutinin (PHA). Likewise, CAT expression was present at only low levels in circulating monocytes, but higher levels of CAT were observed in macrophages grown in the presence of various cytokines (CSF-1, GM-CSF, IL-1 alpha, IL-4, and IL-2). Furthermore, Langerhans cells recovered from skin showed higher levels of CAT activity than those observed in other cells of monocyte-macrophage lineage. These results indicate that LTR-CAT expression in cells of monocyte-macrophage lineage may increase in proportion to the degree of differentiation of these cells. These animals may be useful in the study of cell-specific determinants of LTR-directed gene activity and may serve to identify exogenous cofactors that promote the progression of HIV-related disease in vivo.

Animals↗
PubMed1989

Transgenic mammals and biotechnology.

Biotechnology has begun to realize the enormous potential of transgenic technology: mice with human genes that produce human proteins of therapeutic value in their milk, pigs that express bovine genes that help them gain weight and lose backfat, animals with engineered gene defects that mimic human genetic diseases.

Animals↗
PubMed1989

Perturbed development of the mouse lens by polyomavirus large T antigen does not lead to tumor formation.

To study how the oncogenic process may involve effects on differentiation, we overexpressed an immortalizing oncogene in a developing tissue in transgenic mice. By use of a gene fusion of the alpha A-crystallin promoter to the viral immortalizing oncogene, polyoma large T antigen (PyLT), we created transgenic mice that express PyLT specifically in ocular lens. Expression of large T antigen during embryonic development led to a perturbation in lens development, specifically, an interference with the normal program of fiber cell differentiation. This resulted in microphthalmia, which persisted throughout the life of the animal. Histological analysis revealed impairment of cell elongation, denucleation, and mitotic senescence in both primary and secondary fiber cell differentiation. Strikingly, there was no evidence for hyperplasia or for tumor development in vivo, unlike the consequences of many immortalizing oncogenes on tissues in other transgenic mice. In vitro, however, the developmentally perturbed cells derived from the transgenic lens showed high proliferative capacity. Our results suggest that a primary effect of aberrant expression of an immortalizing gene is an interference with normal tissue development; however, this interference may not necessarily induce proliferation or lead to tumor formation.

Animals↗
PubMed1989

Functional human CD4 protein produced in milk of transgenic mice.

The soluble form of human CD4, an HIV receptor molecule first detected on the surface of T cells, binds glycoprotein gp120, a coat protein of human immunodeficiency virus, and has potential value for the treatment of AIDS. As a first step toward providing the necessary quantities of this protein at an affordable price we report here on the production of functional, soluble human CD4 in transgenic mice. In these animals, a regulatory region derived from a murine gene encoding the whey acidic protein directs synthesis of human CD4 protein to the mammary gland of lactating animals where it is secreted into milk.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Differentiation and oncogenesis: phenotypically distinct lens tumors in transgenic mice.

We report on two lines of transgenic mice that express a murine alpha A-crystallin/SV40 tumor antigen fusion gene in the eye lens. The alpha T1 line develops fast growing, poorly differentiated lens tumors, whereas the alpha T2 line produces lens tumors that are slow growing and well differentiated. There is a striking difference between these two lines in the temporal and spatial patterns of tumor antigen expression during initial lens development. In the alpha T1 line, the transgene is expressed very early in development in most lens cells, and no primary fiber differentiation takes place. In the alpha T2 line, transgene expression occurs after primary fiber formation has been initiated, and is restricted to differentiating fiber cells. The anterior epithelium from both alpha T lines undergoes normal development and remains morphologically normal until after birth, although in alpha T1 mice, these anterior cells produce considerable amounts of SV40 tumor antigens. This suggests that the state of differentiation of the lens cell plays an important role in its response to oncogene products.

Animals↗
PubMed1988

Expression and amplification in transgenic mice of a polyoma virus mutant regulatory region.

Two hybrid gene constructs consisting of wild-type and mutant polyoma regulatory regions fused to a bacterial reporter gene were inserted in the mouse germline. Both transgenes were expressed in a large number of different organs. However, marker gene expression controlled by the polyoma wild-type regulatory region was not detectable in the early embryo and remained low throughout the life of the animal while expression controlled by the polyoma F9-1 mutation was detectable in blastocysts and was significantly higher at later stages of development. The F9-1 hybrid gene was also amplifiable when large T-antigen was supplied in trans to mice or to kidney cells derived from these transgenic mice. Amplification resulted in the appearance of several hundred copies of episomal transgenes and a marked increase of marker gene RNA and protein. Our results suggest that the F9-1 mutation does not alter the target spectrum of gene expression in vivo but does create a more efficient enhancer element in the polyoma early control region. Transgene amplification based upon use of the polyoma regulatory elements may be a means of increasing expression of genes in transgenic mice.

Age Factors↗
PubMed1988

Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus.

Indirect immunofluorescence shows that purified T7 RNA polymerase, when microinjected into monkey kidney (Vero) cells, localizes predominantly in the cytoplasm. To direct active T7 RNA polymerase to the nucleus, we first created unique restriction sites at two locations within the cloned gene for T7 RNA polymerase, T7 gene 1 and then inserted into these sites a 36-bp synthetic nucleotide sequence encoding the SV40 T antigen nuclear location signal. Insertion of the nuclear location signal between codons 10 and 11 of T7 RNA polymerase has only minimal effect on transcription activity in Escherichia coli, but its insertion four codons from the C terminus abolishes activity. Fusion proteins having only foreign codons ahead of codon 11 also have transcription activity in E. coli. Such fusion proteins can be expressed transiently from plasmids microinjected into monkey cells, using SV40 expression signals, and detected by immunofluorescence. A fusion protein containing a nuclear location signal localized predominantly in the nucleus whereas those which lack the signal localize predominantly in the cytoplasm. Ability to direct T7 RNA polymerase to the nucleus may be an advantage in attempting to make this enzyme useful for selective transcription in eukaryotic cells.

Amino Acid Sequence↗
PubMed1988

Gene transactivation mediated by the TAT gene of human immunodeficiency virus in transgenic mice.

Transgenic mice were generated carrying either the long terminal repeat of Human Immunodeficiency Virus fused to the bacterial chloramphenicol acetyl transferase reporter gene or a control element of the murine alpha A crystallin gene fused to the tat gene of human immunodeficiency virus. By crossing these two strains, progeny were obtained which carried both transgenes. The bacterial reporter gene was specifically transactivated in the eyes of these animals.

Acetyltransferases↗
PubMed1988

Perturbations of lens development in the transgenic mouse.

Interference with specific features of cell growth and differentiation in vivo offers rare insights into the complex mechanism of cell proliferation in the developing organism. We have inserted in the germ line of mice transgenes which direct dominant changes in cell development and proliferation to the lens, a tissue exquisitely suited for in vivo analysis. Lens-specific expression of SV40 T antigens results in malignant lens tumors or phakomas, that of a murine c-mos protooncogene in a characteristic defect of lens fiber differentiation.

Animals↗
PubMed1988

A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice.

Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.

Animals↗
PubMed1988

Antisense Myc sequences induce differentiation of F9 cells.

Down-regulation of Myc expression is the earliest documented change in gene expression in retinoic acid-induced differentiation of murine F9 teratocarcinoma cells. F9 cells transfected with plasmids expressing antisense Myc sequences under control of the simian virus 40 (SV40) early promoter exhibit a decrease in Myc protein. The result of this decrease is the spontaneous differentiation into cells that resemble retinoic acid-treated F9 cells as judged by plasminogen activator assays. In contrast, when F9 cells are transfected with a plasmid expressing Myc under control of the SV40 early promoter, resulting cell clones are resistant to differentiation by retinoic acid as shown by the lack of induction of plasminogen activator. These results suggest that down-regulation of Myc is sufficient and necessary for F9 cell differentiation.

Animals↗
PubMed1988

Prenatal lethality in a transgenic mouse line is the result of a chromosomal translocation.

We have produced a line of transgenic mice that is characterized by prenatal lethality. These mice bear a chimeric plasmid containing the long terminal repeat of the Rous sarcoma virus linked to the coding region of the chloramphenicol acetyltransferase gene (pRSV-CAT). Mice heterozygous for the pRSV-CAT integration site are semisterile, producing litters approximately equal to 40% of the average size when crossed to normal mice. Approximately 50% of the progeny from such a cross bear the pRSV-CAT sequences and also produce litters of smaller size. An analysis of embryogenesis revealed that normal numbers of embryos implanted, but 60% failed to develop past day 7. Eight other independent transgenic lines containing RSV-CAT show no evidence of embryonic lethality; thus, it is unlikely that the defect observed is due to the direct effects of RSV-CAT expression. We have found that carrier mice bear a reciprocal translocation between chromosomes 6 and 17, T(6A2-6A3;17D-17E1), that can explain the apparent dominant embryonic lethality seen in this line. The site of integration has been localized by in situ hybridization at or near the translocation breakpoint in one of the translocated chromosomes (6(17)). Because the foreign DNA is present in one of the translocated chromosomes, we propose that this rearrangement was elicited by the introduction of foreign DNA.

Animals↗
PubMed1988

Expression of homeobox gene Hox 1.1 during mouse embryogenesis.

Many of the genes controlling segmentation and pattern formation in Drosophila contain a conserved 183 bp sequence known as the homeobox. Homeobox sequences have been found in a range of metazoan species, including the vertebrates mouse and man. This striking conservation suggests that homeobox genes may play a fundamental role in developmental processes. If this is the case then it might be expected that vertebrate homeobox genes will be differentially expressed during embryogenesis and that the timing of their expression will coincide with major morphogenetic events. Here the spatial and temporal patterns of expression of murine homeobox genes will be explored, concentrating on the Hox 1.1 gene as an example. Using in situ hybridization to localize RNA transcripts, it has been found that Hox 1.1 is expressed in a region-specific manner during the formation and differentiation of the embryonic anteroposterior axis. Although striking patterns of expression of Hox 1.1 and other homeobox genes are seen in overtly segmented structures of the embryo (i.e. somites, prevertebral elements, neural tube and dorsal spinal ganglia) expression is also seen in tissues with no obvious segmental origin. The results suggest that homeobox genes probably do not play an exclusive role in segmentation in vertebrates, but are consistent with a role in the assignment of positional identity along the axis of the embryo.

Animals↗
PubMed1987

Oncogenesis of the lens in transgenic mice.

Neoplastic tumors of the ocular lens of vertebrates do not naturally occur. Transgenic mice carrying a hybrid gene comprising the murine alpha A-crystallin promoter (-366 to +46) fused to the coding sequence of the SV40 T antigens developed lens tumors, which obliterated the eye cavity and even invaded neighboring tissue, thus establishing that the lens is not refractive to oncogenesis. Large-T antigen was detected early in lens development; it elicited morphological changes and specifically interfered with differentiation of lens fiber cells. Both alpha- and beta-crystallins persisted in many of the lens tumor cells, while gamma-crystallin was selectively reduced. Accessibility, characteristic morphology, and defined protein markers make this transparent epithelial eye tissue a potentially useful system for testing tumorigenicity of oncogenes and for studying malignant transformation from its inception until death of the animal.

Animals↗