Biomedical subjects

R E Hammer

Publications and source records attributed to R E Hammer.

At least 127 records · Page 7Linked to original sources

PubMed1990

The expression of influenza virus hemagglutinin in the pancreatic beta cells of transgenic mice results in autoimmune diabetes.

Insulin-dependent diabetes mellitus results from the autoimmune destruction of the insulin-producing beta cells of the pancreatic islets. The target antigen(s) involved in this immunopathological process has not been identified. Our strategy was to determine whether expression of a novel surface antigen by murine pancreatic beta cells would result in insulin-dependent diabetes mellitus. We have generated lines of transgenic mice (RIP-HA) that express the hemagglutinin of the A/Japan/305/57 strain of influenza virus on their insulin-producing beta cells. Hyperglycemia developed in mice derived from all three founders at a frequency varying from 13% to 27%, and was associated with lymphocytic infiltration of the islets and a humoral response against beta cell antigens, including hemagglutinin. These results suggest that the RIP-HA mice should provide a useful system in which to study the cellular interactions involved in the induction of self-tolerance and autoimmunity.

Animals↗

PubMed1990

Effect of the expression of a hepatocyte-specific MHC molecule in transgenic mice on T cell tolerance.

A hybrid murine class I gene, Q10/L, was injected into C3H/HeJ fertilized ova to produce transgenic (TG) mice. This fusion gene contained 414 bp of Q10 promoter sequences which was sufficient to direct liver-specific expression in two lines of animals. Animals from these lines did not have Q10/L mRNA in 10 nonhepatic tissues examined including thymus, spleen, and bone marrow. The ontogeny of Q10/Ld expression in both liver and yolk sac paralleled expression of endogenous Q10. Analysis of liver cells from these lines by flow cytometry and immunofluorescence demonstrated the presence of the Q10/L Ag solely on hepatocytes. TG animals showed no signs of hepatic disease as evidenced by an absence of cellular infiltrates in the liver and a normal profile of serum enzymes that are elevated in association with hepatic disease. When spleen cells from TG animals were cocultured with splenocytes that express Ag cross-reactive with Q10/L, CTL were generated that recognized and lysed L cells which express Q10/L. However, the extent of lysis was less than that generated from non-TG control littermates. That these cross-reactive T cells were physiologically significant was demonstrated by adoptive transfer of in vivo primed T cell enriched spleen cells which produced a mononuclear infiltration of the liver of TG recipients. However, inoculation of Q10/L L cells or splenocytes expressing Q10/L cross-reactive Ag into TG mice did not induce cellular infiltration or overt hepatic disease. Whereas inoculation of normal C3H mice with these cells led to priming of Q10/L reactive CTL, anti-Q10/L CTL could not be primed in TG mice. This suggests that Ag expression solely on hepatocytes can lead to inactivation of specific CTL clones and thus account for the observed in vivo tolerance.

Age Factors↗

PubMed1990

Relative growth of the skull and postcranium in giant transgenic mice.

Cross-sectional allometric growth patterns of the cranial and postcranial skeleton were compared between giant transgenic (MT-rGH) mice and their normal littermate controls. Body weights, external body dimensions, and a series of cranial and postcranial linear dimensions of the skeleton were determined for samples of known age. Comparative bivariate and multivariate allometric analyses were completed in order to determine whether (1) the larger transgenic mice differed significantly from the normal controls in aspects of body and skeletal proportions, and (2) any such proportion differences resulted from general allometric effects of overall weight or skeletal size increase. Results demonstrate that the transgenic mice do exhibit significantly different body and skeletal proportions than normal control adults. Allometric comparisons of the skeletal dimensions relative to body weight reveal similar coefficients of growth allometry but several differences in gamma-intercept values in the transgenic vs. control groups. The comparisons among the skeletal dimensions of the skull and postcranium generally reveal the sharing and differential extension of common growth allometries in the two groups. Thus, the elevated levels of growth hormone (GH) and insulin-like growth factor I (IGF-I) in the transgenic mice appear to result in increased overall growth for the various skeletal elements, but in the relative proportions determined by intrinsic growth controls within that system.

Animals↗

PubMed1990

Peripheral tolerance in mice expressing a liver-specific class I molecule: inactivation/deletion of a T-cell subpopulation.

We previously demonstrated that C3H/HeJ transgenic (TG) mice that express a laboratory-engineered class I molecule, Q10/L, exclusively on liver parenchymal cells show no evidence of hepatic disease even after deliberate immunization. Nevertheless, these animals demonstrate cytotoxic T-lymphocyte (CTL) activity specific for Q10/L, although it is less than that obtained from non-TG littermates. We now show that this decrease in CTL activity is not a reflection of a decrease in precursors, since both TG and normal animals have similar numbers. When non-TG C3H mice are primed with H-2Ld and H-2Kbm1 antigens, which extensively crossreact with Q10/L, their specific in vitro CTL activity directed against H-2Ld, H-2Kbm1, and Q10/L is increased 10- to 20-fold, as expected. Although primed TG mice show similar increases in in vitro CTL activity directed against H-2Ld and H-2Kbm1, they display no increase in anti-Q10/L activity. Whereas anti-H-2Ld spleen cells from non-TG mice readily generate CTL lines and clones specific for H-2Ld and Q10/L, TG cells give rise to anti-H-2Ld lines or clones only. These data indicate that the tolerance in TG mice is accounted for by the inactivation or deletion of an important CTL subpopulation having the capability of recognizing the peripheral antigen in situ. To determine whether tolerance would persist in the absence of Q10/L, TG cells were transferred into non-TG recipients. Three weeks later Q10/L-specific lytic activity generated in in vitro bulk cultures remained reduced compared to non-TG cells, indicating that the tolerant phenotype was stable during this interval.

Animals↗

PubMed1990

Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice.

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.

Animals↗

PubMed1990

Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice.

Lp(a) lipoprotein purified from human plasma bound with high affinity to isolated bovine LDL receptors on nitrocellulose blots and in a solid-phase assay. Lp(a) also competed with 125I-LDL for binding to human LDL receptors in intact fibroblasts. Binding led to cellular uptake of Lp(a) with subsequent stimulation of cholesterol esterification. After intravenous injection, human Lp(a) was cleared slowly from the plasma of normal mice. The clearance was markedly accelerated in transgenic mice that expressed large amounts of LDL receptors. We conclude that the covalent attachment of apo(a) to apo B-100 in Lp(a) does not interfere markedly with the ability of apo B-100 to bind to the LDL receptor and that this receptor has the potential to play a major role in clearance of Lp(a) from the circulation of intact humans.

Adult↗

PubMed1990

Expression and performance in transgenic pigs.

Recent research clearly shows that fusion genes can be microinjected into a pronucleus of an ovum and integrate into the pig genome. Animals with such fusion genes are called 'transgenic'. The percentage of injected ova that developed into transgenic pigs varied among experiments from 0.31% to 1.73%. The percentage of transgenic pigs that expressed the fusion gene ranged from 17% to 100%. Eleven different regulatory sequences have been used for fusion genes transferred into pigs. Some of these regulatory sequences directed strong gene expression, but control over level of expression was inadequate. Other regulatory sequences directed weak expression, but imparted only brief spikes of induced expression. The predominant gene coding sequences transferred were for growth-related hormones. Elevation of growth hormone (GH) in expressing transgenic pigs enhanced plasma concentrations of insulin-like growth factor-I (IGF-I), insulin, and glucose, improved feed efficiency about 15%, and markedly reduced subcutaneous fat compared to nontransgenic siblings. Growth rate was enhanced in some transgenic GH pigs but not in others, possibly due to dietary limits. The 'over-expression' of GH was detrimental to the general health of most transgenic pigs. The most prevalent problems were lethargy, lameness, and gastric ulcers. Gilts that expressed foreign GH genes were anoestrous. Boars that expressed foreign GH genes lacked libido, but their semen was fertile when used by artificial insemination. Six different fusion genes have been transmitted from transgenic founders to progeny. Most of the transgenic pigs that produced progeny transmitted the fusion gene as an autosomal dominant trait to about half of their progeny. Regulatory sequences that will permit full control of gene expression must be developed before the full potential of gene transfer in pigs can be realized.

Animals↗

PubMed1990

Insertion, expression and physiology of growth-regulating genes in ruminants.

Transgenic sheep with elevated concentrations of circulating growth hormone (GH) were produced by microinjecting recombinant DNA into pronuclei of zygotes. The transgenes were fusion genes of non-GH promoters with coding sequences of various growth hormone genes including human, ovine or bovine. In addition, sheep transgenic with the human growth hormone releasing factor gene were produced. Non-GH promoters for fusion genes allowed novel regulation of GH production in ectopic tissues, including the kidney, liver and gut. Elevated levels of GH profoundly altered plasma IGF-1 without significantly altering rate of growth or feed efficiency. Carcass composition was altered with reduced fat. Elevated GH induced diabetes, resulting in death by 1 year of age. These studies indicate the need for improved regulation of inserted genes or investigation of alternative systems, such as GH receptors, to improve growth using the transgenic approach in ruminants.

Animals↗

PubMed1990

Integration, expression and germ-line transmission of growth-related genes in pigs.

We have produced transgenic pigs that harbour structural genes for bovine and human growth hormone (bGH and hGH) ligated to a mouse metallothionein-I (MT) promoter, human growth hormone-releasing factor (hGRF) ligated to the MT or mouse albumin (ALB) promoter, and human insulin-like growth factor-I (hIGF-I) ligated to MT promoter. From 0.31 to 1.03% of microinjected ova developed into transgenic pigs with the various fusion genes. Foreign GH was present in plasma of 61% of the MT-hGH and 89% of the MT-bGH transgenic pigs. Two of 7 pigs with MT-hGRF and all 3 ALB-hGRF transgenic pigs had high concentrations of GRF in their plasma, but plasma concentrations of porcine GH (pGH) were not higher in GRF transgenic pigs than in littermate control pigs. In contrast, plasma concentrations at birth ranged from 3 to 949 ng hGH/ml for MT-hGH transgenic pigs and 5 to 944 ng bGH/ml for MT-bGH transgenic pigs. Presence of the foreign GH depressed endogenous pGH to non-detectable levels. In MT-bGH transgenic pigs, plasma IGF-I was elevated more than 2-fold, plasma glucose was elevated about 30 mg/dl, and plasma insulin was 20-fold higher than in littermate or sibling control pigs. Two lines of pigs expressing the MT-bGH transgene gained 11.1% and 13.7% faster, and were 18% more efficient in converting feed to body weight gain than were sibling control pigs. Expression of the MT-bGH transgene caused a marked repartitioning of nutrients from subcutaneous fat into other carcass components, including muscle, skin, bone and certain organs. The persistent excess hGH or bGH in transgenic pigs was detrimental to general health; lameness, lethargy and gastric ulcers were the most prevalent problems. Gilts that expressed the hGH or bGH transgenes were anoestrous. Germ-line transmission was obtained in 4 of 5 expressing transgenic boars and 4 of 5 nonexpressing transgenic boars and gilts. From 2% to 73% of progeny inherited a transgene from founder transgenics. All transgenic progeny of MT-hGH, MT-bGH and MT-hGRF founder males expressed the transgene if their sire also expressed the gene. The concentration of bGH or hGH in plasma of transgenic progeny was similar to the concentration present in the founder transgenic.

Animals↗

PubMed1989

Immunoglobulin kappa gene expression after stable integration. II. Role of the intronic MAR and enhancer in transgenic mice.

MOPC41 immunoglobulin kappa gene constructs have been stably introduced into the mouse germ line to investigate the effects of deleting the conserved intronic sequences on gene expression. Intact kappa genes containing 1.5 kilobase pairs of upstream and 8.5 kilobase pairs of downstream flanking sequences were highly expressed tissue-specifically, raising the total level of kappa mRNA in spleens severalfold in most transgenic animals. This high expression was often accompanied by marked suppression of endogenous kappa gene activity. Transgenes containing a deletion of the matrix association region (MAR) or both the MAR and enhancer were expressed tissue-specifically at mean levels only 2- and 3-fold lower, respectively, than that of intact transgenes. Therefore, while the intronic enhancer and MAR appear to play a quantitative role in gene expression, these sequences are not absolutely essential for transcriptional activation of rearranged kappa genes in a normal developmental environment.

Animals↗

PubMed1989

Metallothionein-vasopressin fusion gene expression in transgenic mice. Nephrogenic diabetes insipidus and brain transcripts localized to magnocellular neurons.

Arginine vasopressin (AVP) is a potent neuroactive and vasoactive nonapeptide encoded in and processed from a precursor, preproarginine vasopressin-neuro-physin II (preproAVP-NPII). To study the physiologic consequences of a genetic model of chronic hypervasopressinemia transgenic mice were produced by introduction of a mouse metallothionein-rat-ppAVP-NPII fusion gene into the germ line of mice. Three stable transgenic pedigrees were analyzed through several generations. Levels of immunoreactive AVP and neurophysin (NP) in sera, livers, kidneys, intestines, pancreas, and brains were markedly elevated. Chromatographic analyses showed sera levels of approximately 500 pg/ml (normal 0-20 pg/ml) of authentic AVP non-apeptide and serum osmolalities were elevated, 315.4 +/- 1.4 mosm/liter (control, 307.3 +/- 1.1), consistent with a state of mild nephrogenic diabetes insipidus. Brain levels of immunoreactive AVP in transgenic mice were 3-4-fold elevated 145 +/- 15 ng/g versus 31 +/- 7 (controls). Although immunoreactive AVP in livers and intestines, and to some extent kidneys, consisted predominantly of unprocessed precursors, in brain and pancreas greater than 90% of AVP consisted of processed bioactive nonapeptide, as determined by chromatography and measurements of cAMP-generation in LLC-PK1 cells. Immunocytochemistry localized immunoreactive AVP to the exocrine pancreas and to the magnacellular neurons (SON and PVN) of the hypothalamus. Expression of the fusion gene in the hypothalamus was further demonstrated by Northern analyses of fusion gene specific transcripts and in situ histohybridization. Although the fusion gene contained only 35 base pairs of 5'-flanking DNA of the ppAVP-NPII gene, a tentative neuronal cell-specific expression element, -17GCCCAG-CC-10 resides in this sequence and may confer neuron-specific expression to the fusion gene.

Animals↗

PubMed1989

Genetic engineering of livestock.

Genetic engineering of livestock is expected to have a major effect on the agricultural industry. However, accurate assessment of the consequences of transgene expression is impossible without multigenerational studies. A systematic study of the beneficial and adverse consequences of long-term elevations in the plasma levels of bovine growth hormone (bGH) was conducted on two lines of transgenic pigs. Two successive generations of pigs expressing the bGH gene showed significant improvements in both daily weight gain and feed efficiency and exhibited changes in carcass composition that included a marked reduction in subcutaneous fat. However, long-term elevation of bGH was generally detrimental to health: the pigs had a high incidence of gastric ulcers, arthritis, cardiomegaly, dermatitis, and renal disease. The ability to produce pigs exhibiting only the beneficial, growth-promoting effects of growth hormone by a transgenic approach may require better control of transgene expression, a different genetic background, or a modified husbandry regimen.

Agriculture↗

PubMed1989

Production of transgenic sheep with growth-regulating genes.

Pronuclei of fertilized sheep ova were injected with fusion genes consisting of the mouse metallothionein-I promotor/regulator ligated to either the structural gene for bovine growth hormone (mMTbGH) or to a minigene for human growth hormone-releasing factor (mMThGRF). From a total of 842 sheep ova injected with mMTbGH and transferred into recipient ewes, 47 lambs were born. Two of the lambs were transgenic with mMTbGH, and both had bGH mRNA present in liver, kidney, and gut. In one lamb, plasma growth hormone was as high as 700 ng/ml. From a total of 435 sheep ova injected with mMThGRF and transferred to recipients, 54 lambs were born and 9 fetuses were collected. Nine of the 63 had integrated the mMThGRF gene. One of the nine had high concentrations of immunoassayable hGRF in its plasma and high variable plasma concentrations of ovine growth hormone. The lamb that expressed the hGRF gene did not release GH in response to an hGRF challenge. Four of five fetal offspring of a nonexpressing mMThGRF transgenic ram also contained the mMThGRF gene and, like the sire, failed to express the gene as determined by either liver hGRF mRNA or by plasma hGRF. Growth of the single transgenic lamb expressing hGRF was similar to control lambs. These studies demonstrate efficient introduction of genes into the sheep genome and indicate that transgenes are expressed and heritable.

Animals↗

PubMed1989

Differential requirements for cell-specific elastase I enhancer domains in transfected cells and transgenic mice.

The 134-bp enhancer region of the pancreatic elastase I gene is sufficient to direct pancreatic acinar cell-specific transcription in transgenic mice and in transfected cells in culture. Ten-base-pair scanner mutations in three separate enhancer domains that inactivate enhancer function in transfected pancreatic cells in culture have no significant effect in transgenic mice. Because any pair of the three domains is sufficient to direct pancreas-specific expression in mice, no one domain is required for pancreas-specific transcription. Disruption of any two domains does inactivate the enhancer function in transgenic mice. Therefore, the elastase I enhancer domains essential for function in transfected cells in culture are not essential in animals but have a redundant function not apparent in transfected cells. This redundant function is not because of the particular acinar cell line used for transfections, the nature of the reporter gene, or the state of integration of the foreign test gene. We conclude that a trans-acting transcription factor or a modification of a factor(s) present in pancreatic cells of an animal is absent in pancreatic acinar cell lines.

Animals↗

PubMed1989

Histopathology associated with elevated levels of growth hormone and insulin-like growth factor I in transgenic mice.

Serum levels of GH and insulin-like growth factor I (IGF-I) were genetically increased to investigate the physiological activities of these proteins. Lines of mice expressing chimeric genes composed of bovine GH, human GRF, or human IGF-I coding sequences fused to the mouse metallothionein I promoter were examined for consequences of chronic exposure to high levels of these peptides. Animals with elevated serum levels of GH (either bovine GH or mouse GH) have selective splanchnomegaly coupled with glomerular sclerosis and hepatocellularmegaly. Serum levels of insulin and cholesterol are increased. In contrast (with the exception of selective enlargement of organs), the chronic expression of IGF-I results in a different pattern of abnormalities. These findings suggest that the pathogenesis of GH-related disorders is not mediated solely by IGF-I.

Animals↗

PubMed1989

Expression of human or bovine growth hormone gene with a mouse metallothionein-1 promoter in transgenic swine alters the secretion of porcine growth hormone and insulin-like growth factor-I.

Endocrine profiles were examined in swine that had integrated and expressed a fusion gene consisting of mouse metallothionein-1 (MT) promoter fused to either a human (h) or bovine (b) GH structural gene. Eleven of 18 pigs that had integrated MT-hGH and eight of nine pigs that had integrated MT-bGH expressed the genes. The level of expression varied widely among pigs (14-4551 micrograms/l for MT-hGH and 23-1578 micrograms/l for MT-bGH). The level of expression varied over time within each pig with no general pattern. Concentrations of porcine GH (pGH) were lower in MT-hGH pigs that expressed the gene than in non-expressors or in littermate controls. Insulin-like growth factor-I (IGF-I) concentrations increased with age in all pigs and were raised threefold in pigs expressing either the MT-hGH or MT-bGH genes. Measurement of the foreign GH in samples taken at 15-min intervals failed to reveal any short-term fluctuations in concentration. Administration of hGH releasing factor (GRF) to pigs expressing MT-bGH resulted in attenuated release of pGH compared with that of contemporary controls. Concentrations of bGH did not change after GRF injection. Human and bovine GH expressed in transgenic pigs appear to be biologically active in that they induce IGF-I and suppress endogenous pGH secretion. The failure to find short-term fluctuations and the lack of response to GRF injections are consistent with a non-pituitary and non-GRF regulatable site of production.

Animals↗

PubMed1988

Studies of HLA-B27-associated disease.

Several rheumatic diseases were first shown to be associated with human leukocytic antigen (HLA)-B27 in 1973. Recent developments in understanding this association include the finding that there are at least six variants of HLA-B27 at the molecular level, with no one variant preferentially associated with disease. Detailed studies of the structure of the HLA-B27 molecular family are in progress in several laboratories. Mice expressing HLA-B27 and transmitting it to their offspring (transgenic mice) have been produced and are being studied for their response to bacteria that are known to trigger reactive arthritis in B27+ humans. A particular restriction fragment length polymorphism was recently claimed to be a genetic marker for an additional risk factor in ankylosing spondylitis, but two other laboratories have failed to confirm this finding.

Animals↗

PubMed1988

HLA-B27 in inbred and non-inbred transgenic mice. Cell surface expression and recognition as an alloantigen in the absence of human beta 2-microglobulin.

A gene encoding the H chain of the human class I MHC Ag HLA-B27 was introduced into the germ lines of inbred C57BL/6 (B6) and non-inbred (B6 X SJL/J) F2 mice. By immunofluorescence and flow cytometry, the HLA-B27 gene product was expressed on lymphoid cells at levels comparable to the endogenous H-2b and H-2s class I MHC molecules. In both primary and secondary MLC between responder spleen cells from non-transgenic (B6 X SJL/J) F1 mice and transgenic stimulator cells, CTL were generated that specifically lysed mouse L cell (H-2k) or human B cell targets expressing HLA-B27, and this lysis thus appeared largely unrestricted by H-2. These results indicate that transgenic mice express a functional HLA-B27 gene product on cell surfaces in the absence of the human beta 2-microglobulin gene. These transgenic mice promise to be a valuable resource in the investigation of the unique role of HLA-B27 in inflammatory human disease.

Animals↗