Biomedical subjects

P A Overbeek

Publications and source records attributed to P A Overbeek.

18 recordsLinked to original sources

PubMed1992

Expression of yeast hexokinase in pancreatic beta cells of transgenic mice reduces blood glucose, enhances insulin secretion, and decreases diabetes.

It has been proposed that endogenous hexokinases of the pancreatic beta cell control the rate of glucose-stimulated insulin secretion and that genetic defects that reduce beta-cell hexokinase activity may lead to diabetes. To test these hypotheses, we have produced transgenic mice that have a 2-fold increase in hexokinase activity specific to the pancreatic beta cell. This increase was sufficient to significantly augment glucose-stimulated insulin secretion of isolated pancreatic islets, increase serum insulin levels in vivo, and lower the blood glucose levels of transgenic mice by 20-50% below control levels. Elevation of hexokinase activity also significantly reduced blood glucose levels of diabetic mice. These results confirm the role of beta-cell hexokinase activity in the regulation of insulin secretion and glucose homeostasis. They also provide strong support for the proposal that reductions in beta-cell hexokinase activity can produce diabetes.

Animals↗

PubMed1992

5' flanking sequences of the murine adenosine deaminase gene direct expression of a reporter gene to specific prenatal and postnatal tissues in transgenic mice.

Adenosine deaminase (ADA), an enzyme of purine metabolism, is highly expressed in four tissues of the mouse: the maternal decidua, the fetal placenta, the keratinizing epithelium of the upper alimentary tract (tongue, esophagus, and forestomach), and the absorptive epithelium of the proximal small intestine. ADA is produced at relatively low levels in all other tissues. To identify genetic elements that direct appropriate prenatal and postnatal expression of the ADA gene, a segment of DNA including the ADA promoter and 6.4 kilobases of the adjacent 5' flanking region was tested for the ability to direct the expression of a reporter gene in transgenic mice. In seven lines of transgenic mice studied, this construct directed high levels of reporter gene expression in the placenta and forestomach and exhibited correct developmental regulation in these tissues. This construct failed to direct significant reporter gene expression to either the maternal decidua or the proximal small intestine. Thus, different gene regulatory elements are required to target high expression to the four tissues characterized by high levels of ADA.

Adenosine Deaminase↗

PubMed1992

Photoreceptor degeneration induced by the expression of simian virus 40 large tumor antigen in the retina of transgenic mice.

Expression of the viral oncogene encoding the simian virus 40 (SV40) large tumor antigen (T antigen) typically promotes tumorigenesis in mammalian cells. To generate transgenic mice that express T antigen in rod photoreceptors, a chimeric construct consisting of a mouse opsin promoter fragment fused to the coding region of SV40 T antigen was generated. Expression of T antigen in the transgenic retina began at early stages of postnatal development concomitant with expression of endogenous opsin. Instead of inducing hyperplasia or tumor formation, T-antigen expression caused a rapidly progressing photoreceptor degeneration. The degeneration was accompanied by sustained DNA synthesis in photoreceptor cells, as evidenced by incorporation of [3H]thymidine and by the appearance of mitotic figures at postnatal day 10, a stage when nontransgenic photoreceptor cells are postmitotic and quiescent. Although transgenic photoreceptor cells undergo S phase and enter mitosis, the consequences of T-antigen expression are not proliferation and tumorigenesis but proliferation and cell death.

Animals↗

PubMed1992

Photoreceptor-specific activity of the human interphotoreceptor retinoid-binding protein (IRBP) promoter in transgenic mice.

In order to define the cellular specificity of the interphotoreceptor retinoid-binding protein (IRBP) promoter in the retina, we linked the human IRBP promoter to the beta-galactosidase (lacZ) gene and made five lines of transgenic mice. In three of the five transgenic mouse lines, retinas showed positive staining upon incubation with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). Mice from one line (OVE278B) showed positive X-gal staining throughout the retina except for the most peripheral regions. Interestingly, the staining was heterogeneous throughout the retina. Heavily stained regions were interspersed with lightly stained areas. Mice in two other lines showed highly mosaic X-gal staining patterns. Histological examination demonstrated that staining was confined to photoreceptor cells in all three expressing families. Furthermore, electron microscopy showed that the promoter is active in both rod and cone cells. Our results demonstrate that the human IRBP promoter can be used to obtain photoreceptor-specific gene expression in transgenic mice.

Animals↗

PubMed1992

Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter.

We have previously shown that postnatal expression of the viral oncoprotein SV40 T antigen in rod photoreceptors (transgene MOT1), at a time when retinal cells have withdrawn from the mitotic cycle, leads to photoreceptor cell death (Al-Ubaidi et al., 1992. Proc. Natl. Acad. Sci. USA. 89:1194-1198). To study the effect of the specificity of the promoter, we replaced the mouse opsin promoter in MOT1 by a 1.3-kb promoter fragment of the human IRBP gene which is expressed in both rod and cone photoreceptors during embryonic development. The resulting construct, termed HIT1, was injected into mouse embryos and five transgenic mice lines were established. Mice heterozygous for HIT1 exhibited early bilateral retinal and brain tumors with varying degrees of incidence. Histopathological examination of the brain and eyes of three of the families showed typical primitive neuroectodermal tumors. In some of the bilateral retinal tumors, peculiar rosettes were observed, which were different from the Flexner-Wintersteiner rosettes typically associated with human retinoblastomas. The ocular and cerebral tumors, however, contained Homer-Wright rosettes, and showed varying degrees of immunoreactivity to antibodies against the neuronal specific antigens, synaptophysin and Leu7, but not to antibodies against photoreceptor specific proteins. Taken together, the results indicate that the specificity of the promoter used for T antigen and/or the time of onset of transgene expression determines the fate of photoreceptor cells expressing T antigen.

Animals↗

PubMed1991

Retina-specific expression from the IRBP promoter in transgenic mice is conferred by 212 bp of the 5'-flanking region.

IRBP is a photoreceptor-specific glycoprotein that has been suggested as a retinoid carrier in the visual process. Previous research has shown that 1.3 kb of 5'-flanking sequence from the human IRBP gene is sufficient to promote photoreceptor-specific expression of reporter genes in transgenic mice. To define more narrowly the sequences that promote tissue-specific expression, chimeric constructs with shorter promoters were used to generate transgenic mice. The bacterial CAT gene was fused to fragments of 706 bp or 212 bp from the 5' end of the human IRBP gene. Analysis of the three transgenic families bearing the 706 bp IRBP promoter revealed that CAT expression was confined to the neuro-retina and the pineal gland. Analysis of the four transgenic families bearing the 212 bp IRBP promoter revealed the same tissue-specific CAT expression in three families. These results establish that tissue-specific expression of IRBP can be regulated by a short 212 bp promoter which has been conserved between humans and mice.

Amino Acid Sequence↗

PubMed1991

Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro, allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo.

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.

Adenosine Deaminase↗

PubMed1991

Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme.

There are two isozymes of angiotensin-converting enzyme (ACE), one produced by somatic tissues and a smaller protein synthesized by developing spermatozoa (testis ACE). To investigate the molecular control of testis ACE, we generated mice transgenic for a construct containing a putative testis-specific ACE promoter linked to the Escherichia coli reporter gene encoding beta-galactosidase. The transgenic mice express beta-galactosidase protein and RNA only within the testis. Histochemical analysis of the transgenic mice shows co-localization of beta-galactosidase protein and endogenous ACE within elongating spermatozoa. These studies demonstrate that transcription of testis ACE is controlled by a strong intragenic testis-specific promoter that is contained within a 698-base pair fragment immediately upstream from the transcription start site of testis ACE. Characterization of the testis ACE promoter may provide insights into the molecular mechanisms controlling cell stage-specific gene expression in the male germ line.

Animals↗

PubMed1991

Testis structure in the sys (symplastic spermatids) mouse.

Testes of mice with the recessive insertional mutation termed symplastic spermatids (sys) were assessed for structural and developmental abnormalities. Homozygous (sys/sys) males are infertile due to an abnormality in spermatogenesis leading to azoospermia. The major interruption to spermatogenesis occurs when the intercellular bridges that connect round spermatids open prematurely resulting in the formation of symplasts. Symplasts contain as many as 285 nuclei. Development of spermatids within symplasts is arrested just before, or just after, elongation of the spermatid nuclei begins. Symplasts degenerate and appear to be phagocytized by Sertoli cells and by intratubular macrophages. In addition, degeneration of young round spermatids and also spermatocytes occasionally is observed. Spermatocyte degeneration is substantial in some tubules and leaves them depleted of cells other than basal compartment cells. Sertoli cell abnormalities are prominent and include intracellular vacuolation, absence of apical processes surrounding round spermatids, degeneration, and occasional sloughing. Although reduplication and infolding of the basal lamina is also seen, this does not appear as a common phenomenon. The sys phenotype is first manifest in animals between 19 days and 22 days of age. Considerable variability is seen in testis histology of prepubertal animals; some display degenerating pachytene spermatocytes and virtually no Sertoli cell vacuoles, while others display vacuoles without apparent elevated numbers of degenerating spermatocytes. Although this study has not revealed the primary cell type(s) affected by the insertional inactivation event, it is possible that the abnormalities in the Sertoli cells are responsible for germ cell degeneration as it is generally recognized that deficits in the Sertoli cell can result in major germ cell abnormalities but not vice versa.

Animals↗

PubMed1991

Coinjection strategy for visual identification of transgenic mice.

Transgenic mice were generated by coinjection of a dominant marker gene that induces fur and eye pigmentation (a tyrosinase minigene) plus an unrelated DNA construction that has a gamma-glutamyl transferase (gamma GT) promoter linked to a ras oncogene. Mice transgenic for gamma GT-ras could be identified in the first and all subsequent generations by simple visual inspection for pigmentation. Furthermore, the gamma-glutamyl transferase promoter was active in kidney but not skin of the transgenic mice, indicating that the cointegrated DNA was active and independently expressed. These results confirm that the tyrosinase minigene can be used for coinjections to allow rapid visual identification of transgenic mice.

Animals↗

PubMed1991

Use of a simplified single-site PCR to facilitate cloning of genomic DNA sequences flanking a transgene integration site.

We have used a simplified single-site PCR based strategy to isolate genomic DNA flanking the transgenic insert in a line of transgenic mice. The flanking sequences, which were refractory to more conventional cloning techniques, were isolated and characterized within 1 week. This strategy begins with the annealing and ligation of a single-stranded oligonucleotide to recessed 5' ends of restriction endonu-clease-digested, size-selected transgenic DNA. Following denaturation, a second oligonucleotide is used to prime DNA synthesis from a position within the transgenic sequences to the end of the genomic restriction fragment, finally synthesizing a complement of the ligated oligonucleotide sequence. Subsequently, a PCR is initiated which uses primers specific for the transgenic DNA and the newly synthesized DNA complementary to the ligated oligonucleotide. The only prerequisite data are sequence information for the transgenic DNA and Southern information regarding the size(s) of restriction fragments that contain the flanking genomic material. This report demonstrates one utility of this technique--enabling rapid isolation of specific mammalian genomic DNA sequences.

Animals↗

PubMed1991

Dominant dysplasia of the lens in transgenic mice expressing the dbl oncogene.

To study the transforming activity of the dbl oncogene and its effect on normal development in vivo, we linked dbl cDNA to promoters with different cell type specificities and used the constructs to generate transgenic mice. The promoters included the mouse alpha A-crystallin promoter, the rat insulin II promoter, and a mouse metallothionein promoter. We also generated transgenic mice carrying a recombinant cosmid clone that contains the entire dbl gene. Mice with the crystallin promoter construct developed cataracts and expressed the dbl protein in their lenses. The architecture of the lenses suggested a block to the normal pattern of differentiation and elongation of the secondary fiber cells. Mice with the insulin II promoter expressed dbl protein in the pancreas but showed no evidence of diabetes and no apparent pancreatic beta-cell defects. Similarly, mice with the metallothionein promoter expressed dbl protein in heart and testes, but showed no pathologic abnormalities in these tissues even after treatment with heavy metals. However, one family of mice carrying the metallothionein promoter construct showed cataracts and a dramatic fibroblastic dysplasia of the lens. One family with the cosmid-dbl gene showed a nearly identical lenticular dysplasia, but with a slower developmental time course. Thus, although the dbl oncogene did not induce neoplasia in any of the mice studied, it is apparently capable of interfering with the ability of the lens epithelial cells to differentiate into lens fiber cells, and of inducing metaplasia of the epithelial cells into fibroblastic cells.

Animals↗

PubMed1990

Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice.

Albinism, due to a lack of melanin pigment, is one of the oldest known mutations in mice. Tyrosinase (monophenol oxygenase, EC 1.14.18.1) is the first enzyme in the pathway for melanin synthesis, and the gene encoding this enzyme has been mapped to the mouse albino (c) locus. We have used mouse tyrosinase cDNA clones and genomic sequencing to study the albino mutation in laboratory mice. Within the tyrosinase gene coding sequences, a G to C transversion at nucleotide 308, causing a cysteine to serine mutation at amino acid 103, is sufficient to abrogate pigment production in transgenic mice. This same base pair change is fully conserved in classical albino strains of laboratory mice. These results indicate that a conserved mutation in the tyrosinase coding sequences is responsible for the classical albino mutation in laboratory mice, and also that most albino laboratory mouse strains have been derived from a common ancestor.

Albinism↗

PubMed1990

Tissue-specific expression in transgenic mice directed by the 5'-flanking sequences of the human gene encoding interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular protein that has been suggested to participate in the visual process as a carrier for visual retinoids. A chimeric gene composed of the human IRBP promoter fused to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. Analysis of six transgenic families revealed that the CAT gene, concomitant with the endogenous IRBP gene, was expressed primarily in the retina and, to a lesser extent, in the pineal gland. These results establish that a 1.3-kilobase fragment from the 5' end of the human IRBP gene is sufficient to direct transgene expression to a visual subdivision of the central nervous system.

Animals↗

PubMed1990

Symplastic spermatids (sys): a recessive insertional mutation in mice causing a defect in spermatogenesis.

A line of transgenic mice that carries an insertional mutation in a gene essential for spermatogenesis is described. Males homozygous for the transgenic insert are sterile, while female homozygotes and both male and female heterozygotes exhibit normal fertility. Developing spermatids in homozygous males form prominent abnormal multinucleated syncytia (symplasts) and do not complete maturation. In addition, abnormal cytoplasmic vacuolation is commonly seen in Sertoli cells. One flank of the transgenic integration site within the genome has been cloned and used to show linkage between homozygosity for the transgene and the mutant phenotype. The flank maps to mouse chromosome 14 approximately 4 centimorgans proximal to the gene encoding esterase-10 (Es-10). As no other gene that is known to be essential for spermatogenesis has been mapped to this region of the genome and as the mutant phenotype is unique, the transgenic insert appears to affect a previously unidentified gene. We have named the mutation "symplastic spermatids" (sys).

Alleles↗

PubMed1989

Calmodulin-induced early-onset diabetes in transgenic mice.

Calmodulin is implicated as the primary transducer of the calcium signal in pancreatic beta cells, where it is present at very high concentrations. We have produced three lines of transgenic mice carrying a calmodulin minigene regulated by the rat insulin II promoter. Immunohistochemistry and hybridization analyses indicated a 5-fold increase in the content of calmodulin and its mRNA in beta cells. Transgenic mice developed severe diabetes within hours of birth. The diabetes was progressive and characterized by elevated blood glucose and glucagon that coincided with reduced serum and pancreatic insulin. The disease appears to have both secretory and cell destruction components. beta Cell pathology was evident in the diabetic neonate but not in the fetus, suggesting that the early onset of diabetes exacerbated possible toxic effects of excess calmodulin. These animals provide a new diabetes model to evaluate how abnormal calcium homeostasis alters the carefully programmed secretory activity and development of the beta cell.

Aging↗

PubMed1989

Insertional inactivation of the downless gene in a family of transgenic mice.

The mouse downless (dl) gene is a morphogenetic gene that plays a role in dermal-epidermal interaction and regulation of hair follicle induction during fetal development. We report here the identification of a transgenic mouse line with an insertional mutation in the dl gene. The genomic sequences flanking the transgenic insert have been cloned and used as hybridization probes to confirm that the mutant transgenic mice are homozygous for the transgenic insert and that the site of integration lies on mouse chromosome 10. Genomic probes that are close to or within the downless gene are now available and should permit the characterization of a gene that is involved in induction of a specific type of epithelial morphogenesis.

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↗