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At least 307 records · Page 17Linked to original sources

PubMed1997

Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus.

Three different null alleles of the myogenic bHLH gene MRF4/herculin/Myf-6 were created recently. The three alleles were similar in design but were surprisingly different in the intensity of their phenotypes, which ranged from complete viability of homozygotes to complete lethality. One possible explanation for these differences is that each mutation altered expression from the nearby Myf-5 gene to a different extent. This possibility was first raised by the observation that the most severe MRF4 knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of the Myf-5 null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viability correlate with the intensity of rib skeletal defects, and the most extreme version of this rib defect is the hallmark phenotype of Myf-5 null animals. In the present study we tested this hypothesis for the two milder MRF4 alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakest MRF4 knockout allele on one chromosome 10 and a Myf-5 knockout allele on the other chromosome, we found that both of these MRF4 alleles apparently downregulate Myf-5 expression by a cis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viable MRF4 allele, together with intensified rib defects for both MRF4 alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis for cis interactions at the MRF4/Myf-5 locus was further examined by fusing a DNA segment containing the entire MRF4 structural gene, including all sequences deleted in the three MRF knockout alleles, with a basal promoter and a lacZ reporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA. cis-acting interactions between Myf-5 and MRF4 may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos.

Alleles↗

PubMed2004

Role of the BMK1/ERK5 signaling pathway: lessons from knockout mice.

Mitogen-activated protein (MAP) kinase cascades play a central role in mediating extracellular stimuli-induced intracellular signaling during cell activation. The fourth and least studied mammalian MAP kinase pathway, big MAP kinase 1 (BMK1), also known as extracellular signal regulated kinase 5 (ERK5), is activated in response to growth factors and stress. Activation of this signaling pathway has been implicated not only in physiological functions such as cell survival, proliferation and differentiation but also in pathological processes such as carcinogenesis, cardiac hypertrophy and atherosclerosis. In recent years a series of gene-targeted mice lacking components within the BMK1 cascade have been generated, which have enabled us to investigate the role of the BMK1 pathway within different tissues. Analyses of these knockout mice have led to major discoveries in the role of BMK1 signaling in angiogenesis and in cardiac development. Moreover, studies using conditional BMK1 knockout mice, which circumvent the early embryonic lethality of BMK1 knockouts, have unveiled the importance of BMK1 in endothelial survival and maintenance of vascular integrity during adulthood. Here we summarize current understanding of the function of BMK1, as well as include new data generated from a series of tissue-specific BMK1 knockout mice in an attempt to dissect the role of the BMK1 pathway in various cell types in animals.

Animals↗

PubMed2000

Adaptive mechanisms of the cardiovascular system in transgenic mice--lessons from eNOS and myoglobin knockout mice.

Transgenic mice have turned out to be important in the analysis of cardiovascular physiology and pathology. A large number of gene knockout and overexpression models have been generated, including genes involved in blood pressure regulation, cardiac function and hemostasis. In this review we concentrate on two models, the endothelial NO synthase and the myoglobin knockout mice. It will be shown that the genetic approach of gene function analysis in mice not only provides new insight into the actual role of the encoded gene product, but also uncovers possible secondary alterations which compensate for the induced change. In the case of NOS knockout mice, upregulation of other NOS isoforms, induction of signal molecules such as prostaglandins or endothelium-derived hyperpolarizing factor may conserve the vasodilatory potential of NOS deficient vessels. In the case of myoglobin knockout mice, even structural changes may contribute to compensate a loss of gene function as shown by the elevated capillary density, which may enhance the oxygen supply to mitochondria. Thus, results obtained by the analysis of gene function in transgenic animals may differ from acute pharmacological interventions in that they reveal the striking ability of an intact organism to effectively adapt to chronic changes in gene expression.

Adaptation, Physiological↗

PubMed2001

The role of KCNQ1/KCNE1 K(+) channels in intestine and pancreas: lessons from the KCNE1 knockout mouse.

KCNE1 (IsK, minK) co-assembles with KCNQ1 (KvLQT1) to form voltage-dependent K(+) channels. Both KCNQ1 and KCNE1 are expressed in epithelial cells of gut and exocrine pancreas. We examined the role of KCNQ1/KCNE1 in Cl(-) secretion in small and large intestine and exocrine pancreas using the KCNE1 knockout mouse. Immunofluorescence revealed a similar basolateral localization of KCNQ1 in jejunum and colon of KCNE1 wild-type and knockout mice. Electrogenic Cl(-) secretion in the colon was not affected by gene disruption of KCNE1; in jejunum forskolin-induced short-circuit current was some 40% smaller but without being significantly different. Inhibition of KCNQ1 channels by 293B (IC(50) 1 micromol l(-1)) and by IKS224 (IC(50) 14 nmol l(-1)) strongly diminished intestinal Cl(-) secretion. In exocrine pancreas of wild-type mice, KCNQ1 was predominantly located at the basolateral membrane. In KCNE1 knockout mice, however, the basolateral staining was less pronounced and the distribution of secretory granules was irregular. A slowly activating and 293B-sensitive K(+) current was activated via cholinergic stimulation in pancreatic acinar cells of wild-type mice. In KCNE1 knockout mice this K(+) current was strongly reduced. In conclusion intestinal Cl(-) secretion is independent from KCNE1 but requires KCNQ1. In mouse pancreatic acini KCNQ1 probably co-assembled with KCNE1 leads to a voltage-dependent K(+) current that might be of importance for electrolyte and enzyme secretion.

Animals↗

PubMed2000

Development of an intragastric enteral model in the mouse: studies of alcohol-induced liver disease using knockout technology.

The establishment of a continuous intragastric enteral feeding protocol in the rat by Tsukamoto and French was a major development in research of alcohol-induced liver disease. Unlike other models which only produce fat, with this model, inflammation, necrosis, and fibrosis can now be studied. However, much of what has been learned to date involves inhibitors or nutritional manipulation which may not be specific. Knockout technology could avoid these potential problems. Therefore, we have adapted a rat long-term intragastric protocol to the mouse so that the knockout technology can be used to study the mechanism of alcohol-induced liver injury. Reactive free radicals are involved in the mechanisms of early alcohol-induced liver injury; however, the key source of these species remains unclear. Cytochrome P450 (CYP) 2E1 is induced predominantly in hepatocytes by ethanol and could be one source of reactive oxygen species leading to liver injury. On the other hand, NADPH oxidase or xanthine oxidase is also a potent source of free radicals. In studies using CYP2E1 and p47phox (NADPH oxidase-deficient) knockout mice with this enteral model, it was reported that oxidants from CYP2E1 play only a small role in the mechanisms of early alcohol-induced liver injury in the mouse. Further, free radicals from NADPH oxidase in Kupffer cells play an important role in early alcohol-induced liver injury. Thus, this new enteral mouse model using knockout technology will provide a powerful tool in alcohol research.

Animals↗

PubMed2006

Metabolism and hepatic toxicity of flutamide in cytochrome P450 1A2 knockout SV129 mice.

BACKGROUND: Flutamide, a nonsteroidal antiandrogen used for treatment of prostate cancer, causes a temporary increase in transaminase and in some cases severe liver dysfunction. It is dominantly metabolized by cytochrome P450 (CYP) 1A2 into 2-hydroxyflutamide (OH-flutamide), which has stronger antiandrogenic activity without obvious cytotoxicity to cultured hepatocytes. We hypothesized that another subsidiary metabolite might be responsible for induction of hepatotoxicity. METHODS: Flutamide was administered daily to CYP1A2 knockout mice and parental SV129 mice to compare pharmacokinetics and appearance of hepatic toxicity. RESULTS: In the CYP1A2 knockout mice, the plasma concentration of flutamide maintained at a high level and OH-flutamide stayed low; a higher amount of FLU-1, an alternative metabolite of flutamide, was detected in urine. Simple repetitive administration of 800 mg/kg of flutamide for 28 days to CYP1A2 knockout mice did not show abnormal elevation of plasma alanine aminotransferase (ALT). However, after the knockout mice were fed with an amino acid-deficient diet for 2 weeks, which reduced the glutathione (GSH) content to 27% of the initial, administration of 400 mg/kg of flutamide increased ALT to over 200 IU/l and histopathologically moderate hepatitis developed. Since FLU-1 itself did not show cytotoxicity or reduce GSH content in vitro, a further metabolized molecule must cause the hepatotoxicity. CONCLUSIONS: Blockade of CYP1A2 produced an unknown potential hepatotoxic molecule through FLU-1, and GSH might play an important role in diminishing the reactive hepatotoxic metabolite.

Alanine Transaminase↗

PubMed2001

Biosynthesis of proteoglycan in bone and cartilage of parathyroid hormone-related protein knockout mice.

Proteoglycans are suggested to regulate cell adhesion, differentiation and mineralization of hard tissues. In vitro studies have shown that many humoral and local factors regulate proteoglycan synthesis. Among them, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have potent stimulating effects on proteoglycan synthesis. However, the exact role of PTHrP on the biosynthesis and metabolism of proteoglycans during skeletal development is not clear. To clarify this point, we examined bony and cartilaginous explants of newborn mice with disrupted PTHrP alleles. Ribs of homozygous PTHrP-knockout mice and wildtype littermates were dissected into bony and cartilaginous regions and metabolically labeled with [35S]sulfate in culture. Radiolabeled proteoglycans were analyzed by column chromatography. The elution profiles of [35S]-labeled proteoglycan from cartilaginous explants did not differ between homozygous PTHrP-knockout mice and wild-type littermates. However, the amount of labeled proteoglycan in homozygous PTHrP-knockout mice was only 4%-5% that of wild-type littermates. In contrast with cartilaginous explants, the amount of labeled proteoglycans in bony explants did not differ between the two genotypes. Interestingly, besides the common major peak (Kd = 0.10-0.16) observed in the bony explants of both genotypes, a minor peak (Kd = 0.42) was specifically present in homozygous PTHrP-knockout mice. This minor peak was earlier than that of free glycosaminoglycan (GAG) chains, suggesting that the core protein, but not GAG chain, was cleaved in the bony explants of homozygous PTHrP. These findings demonstrate a crucial nonredundant role of PTHrP in the regulation of proteoglycan synthesis and metabolism during skeletal development.

Animals↗

PubMed2005

A novel model of polyhydramnios: amniotic fluid volume is increased in aquaporin 1 knockout mice.

OBJECTIVE: To test the hypothesis that amniotic fluid volume is increased in aquaporin 1 knockout mice. STUDY DESIGN: Transgenic mice deficient in aquaporin 1 protein were generated by targeted gene disruption, as described previously. After a cesarean section was performed, intact, individual gestational sacs were removed from the uterus and weighed. Amniotic fluid volume, osmolality, and fetal and placental weights were determined. Data were analyzed by a 1-way analysis of variance for ranks; Dunn's post hoc test was used to analyze significant trends. RESULTS: Analysis of 16 litters showed 35 wild-type, 52 heterozygote, and 33 aquaporin 1 knockout mice. The knockout mice had a greater volume of amniotic fluid and lower amniotic fluid osmolality than their wild-type and heterozygote counterparts. There were no significant differences in fetal or placental weights among the groups. CONCLUSIONS: Aquaporin 1 null fetuses produce a greater volume of more dilute amniotic fluid. Our findings show that aquaporin 1 water channels in fetal membranes may contribute to amniotic fluid volume regulation. We speculate that idiopathic polyhydramnios may be associated with a deficiency of aquaporin 1 channels in human fetal membranes. Transgenic aquaporin 1 knockout mice provide a unique animal of polyhydramnios.

Amniotic Fluid↗

PubMed2005

STOP knockout and NMDA NR1 hypomorphic mice exhibit deficits in sensorimotor gating.

Schizophrenia is a chronic and debilitating disease which is thought to arise from a neuro-developmental disorder. Both the stable tubule-only polypeptide (STOP) protein and the N-methyl-D-aspartate (NMDA) NR1 subunit are involved in neuronal development and physiology. It has therefore been postulated that transgenic mice lacking either the STOP or the NMDAR1 gene would show a 'schizophrenic-like' phenotype. Here, STOP knockout and NMDA NR1 hypomorphic mice were assessed in a behavioural measure that can be used to detect schizophrenic-like phenotypes: a change in sensorimotor gating, measured through prepulse inhibition (PPI). STOP knockout mice were further assessed in another measure of 'schizophrenic-like behaviour': hyperlocomotion. The PPI deficit exhibited by both the STOP knockout and NMDA knockdown mice could not be reversed by acute treatment with the atyptical antipsychotic, clozapine (1 mg/kg, i.p.) but the hyperlocomotion shown by the STOP knockout mice was reversed with the same acute dose of clozapine.

Acoustic Stimulation↗

PubMed2005

Selective increase of dark phase water intake in neuropeptide-Y Y2 and Y4 receptor knockout mice.

Neuropeptide-Y (NPY) is involved in the regulation of ingestive behaviour and energy homeostasis. Since deletion of the NPY Y2 and Y4 receptor gene increases and decreases food intake, respectively, we examined whether water intake during the light and dark phases is altered in Y2 and Y4 receptor knockout mice. The water consumption of mice staying in their home cages was measured by weighing the water bottles at the beginning and end of the light phase during 4 consecutive days. Control, Y2 and Y4 receptor knockout mice did not differ in their water intake during the light phase. However, during the dark phase Y2 and Y4 receptor knockout mice drank significantly more (46-63%, P<0.05) water than the control mice. The total daily water intake over 24 h was also enhanced. The enhanced water intake during the dark phase was not altered by the beta-adrenoceptor antagonist propranolol or the angiotensin AT1 receptor antagonist telmisartan (each injected intraperitoneally at 10 mg/kg). These data indicate that NPY acting via Y2 and Y4 receptors plays a distinctive role in the regulation of nocturnal water consumption. While beta-adrenoceptors and angiotensin AT1 receptors do not seem to be involved, water intake in Y2 and Y4 receptor knockout mice may be enhanced because presynaptic autoinhibition of NPY release and inhibition of orexin neurons in the central nervous system are prevented.

Adrenergic beta-Antagonists↗

PubMed2006

Brain-derived neurotrophic factor conditional knockouts show gender differences in depression-related behaviors.

BACKGROUND: Indirect evidence suggests that loss of brain-derived neurotrophic factor (BDNF) from forebrain regions contributes to an individual's vulnerability for depression, whereas upregulation of BDNF in these regions is suggested to mediate the therapeutic effect of antidepressants. METHODS: We have tested this hypothesis by generating two independent lines of conditional BDNF knockout mice in which the BDNF gene is deleted selectively in forebrain. RESULTS: We show that male conditional knockouts exhibit hyperactivity but normal depression-related behaviors. In contrast, female conditional knockouts display normal locomotor activity but a striking increase in depression-like behavior. We also demonstrate that loss of BDNF in both male and female mice attenuates the actions of the antidepressant desipramine in the forced swim test. CONCLUSIONS: These gender differences in depression-related behaviors in BDNF conditional knockout mice provide direct evidence for a role of BDNF in depression. The results also support the view that forebrain BDNF may be essential in mediating antidepressant efficacy.

Alleles↗

PubMed2005

Influence of the dopamine D2 receptor knockout on pain-related behavior in the mouse.

We studied the role of the dopamine D2 receptor in physiological regulation of pain-related behavior. The experiments were performed in dopamine D2 receptor knockout mice and in their wild-type controls. Baseline sensitivity to thermal nociception was determined by measuring the response latency in the hot plate at three different stimulus temperatures and by determining the radiant-heat-induced paw withdrawal. Mechanical sensitivity was assessed by determining paw withdrawal responses to stimulation with a calibrated series of monofilaments. Intracolonic capsaicin was used to produce sustained pain-related behavior and referred hypersensitivity to mechanical stimulation. The hot plate response latencies were not significantly different between the dopamine D2 receptor knockout and wild-type animals, although the stimulus temperature-dependent decrease in the response latency was steeper in the wild-type group. The radiant-heat-induced paw withdrawal latency was slightly longer in the knockout animals. The number of capsaicin-induced behavioral responses or the latency to the occurrence of the first capsaicin-induced response was not different between the experimental groups. Dopamine D2 receptor knockout animals were more sensitive to mechanical stimulation of the hindpaws than wild-type animals both in the baseline condition and following development of capsaicin-induced referred hypersensitivity in the hindpaws. The results indicate that dopamine D2 receptors influence baseline nociception in the mouse, although this effect is weak and submodality selective. Additionally, dopamine D2 receptors may contribute to attenuation of referred hypersensitivity caused by sustained nociception.

Animals↗

PubMed2005

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms.

Nitric oxide (NO), which is produced from l-arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two-cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two-cell embryos/mouse collected after fertilization in vivo (P<0.01), but the rate of blastocyst formation from two-cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS-deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P<0.001). While all three types of NOS do not seem to play a significant role in pre-ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.

Animals↗

PubMed2006

Structural and immunocytochemical alterations in eye lens fiber cells from Cx46 and Cx50 knockout mice.

In the current study we describe the changes of overall organization of lens fiber cells in connexin 46 (Cx46) and connexin 50 (Cx50) knockout mice. Morphometric analyses and the application of immunocytochemical techniques revealed that in Cx46 knockout lens (Cx46 -/-), where Cx50 is expressed alone, the postnatal differentiation of secondary fiber cells proceeds faster and is characterized by an increased number of smaller fiber cells. Conversely, in Cx50 knockout mice (Cx50 -/-), the lenticular mass is considerably reduced and characterized by a small number of fiber cells added during the postnatal period. The process of terminal differentiation was impaired and generated larger fiber cells still possessing cytoplasmic organelles. Freeze-fracture and fracture labeling revealed that the junctional assembly, packing organization and topographic interactions between connexons and MP26 differed when Cx46 and Cx50 were co-assembled in the wild-type or expressed separately in the two distinct knockout phenotypes. Filipin cytochemistry provided indirect evidence that Cx46 and Cx50 expressed alone are recruited into different lipid environments. Our results represent the structural proof that interaction of connexins and MP26 contributes to the overall organization of the fiber cells.

Animals↗

PubMed2005

Intrathecally-administered histamine facilitates nociception through tachykinin NK1 and histamine H1 receptors: a study in histidine decarboxylase gene knockout mice.

Intrathecal injection of histamine elicited behavioral responses consisting of scratching, biting and licking in conscious mice. To study the participation of histamine in pain perception, histidine decarboxylase knockout mice were examined for pain threshold by means of three different kinds of noxious stimuli: thermal nociception (hot-plate, tail-flick, and paw-withdrawal), mechanical nociception (tail-pressure), and chemical nociception (formalin test and capsaicin test). Mutant mice lacking histidine decarboxylase showed significantly fewer nociceptive responses to the hot-plate, tail-flick, paw-withdrawal, tail-pressure, formalin and capsaicin tests. Sensitivity to noxious stimuli in the histidine decarboxylase knockout mice was significantly lower when compared to the wild-type mice. The intrathecally-administered histamine (400 pmol) significantly shortened the latency in the histidine decarboxylase knockout mice, but not in the wild-type mice in tail-flick tests. Pyrilamine, a histamine H(1) receptor antagonist, but not ranitidine, a histamine H(2) receptor antagonist, produced inhibition of the induced behavioral responses in the tail-flick test when co-administered with histamine. Sendide, a tachykinin NK(1) receptor antagonist, inhibited histamine-induced nociceptive behavior in the histidine decarboxylase knockout mice. In contrast, the treatment with D-(-)-2 amino-5-phosponovaleric acid (D-APV), an N-methyl-D-aspartate (NMDA) receptor antagonist, did not prevent the induction of the behavioral responses by histamine. These studies substantiate the evidence that nociceptive behavior induced by intrathecal injection of histamine is largely mediated through tachykinin NK(1) and histamine H(1) receptors in the spinal cord.

2-Amino-5-phosphonovalerate↗

PubMed2006

Enhanced contractility of small blood vessels in JNK knockout mice.

The c-Jun N-terminal kinases (JNKs) form a subfamily of the mitogen-activated protein kinases (MAPK). These signalling pathways regulate various processes such as mitosis, cellular differentiation, stress response or apoptosis in multicellular organisms. There is rising evidence about the role of JNKs activities in neurodegenerative and metabolic diseases as well as in immunological disorders. The physiological functions of JNKs, however, remain to be elucidated. Recent data have demonstrated an essential role of JNKs in the cardiovascular system and the regulation of carbon hydrate and glucose metabolism. Therefore, we have investigated the contractility of blood vessels in mice with genetically deleted JNK1, JNK2, JNK3 and JNK2+3 isoforms and their respective wildtypes. The contractility of the isolated segments from A. carotis communis was measured by small blood vessel wire myograph. Contraction induced by 80 mM KCl was significantly increased in arteries from JNK2+3 double knockout compared to controls and single knockouts. The maximal contraction generated by the alpha-agonists phenylephrine or noradrenaline (10 microM) was significantly enhanced in JNK2+3 knockout arteries compared with arteries from the remaining strains. Inhibition of NOS by Nw-nitro-l-arginine did not change the pattern of vasoconstriction, but vasoconstriction by noradrenaline following NOS inhibition was significantly enhanced in the arteries from JNK2+3 double knockout mice. In conclusion, genetic deletion of JNK2+3 in mice results in altered contractility of carotid arteries and this might depend on the function of the smooth muscles rather than on the endothelium. These findings have implications for the long-term treatment with pharmacological JNK inhibitors for neurodegenerative or metabolic diseases such as stroke or diabetes.

Animals↗

PubMed2004

The future of wound healing: pursuing surgical models in transgenic and knockout mice.

BACKGROUND: Transgenic and knockout technologies have made determination of the molecular basis of wound healing possible. But there is no comprehensive or standardized approach to the investigation of wound healing in the mouse. A convention is proposed for assessing the multiple dimensions of wound healing. An approach to phenotyping a transgenic or knockout animal in a reproducible fashion is presented using this convention. STUDY DESIGN: Age- and gender-matched wildtype and knockout mice were characterized using six parameters of wound healing: epithelialization, granulation tissue formation, contraction, tensile strength, angiogenesis, and response to ischemia. Six surgical (four standard and two impaired) models were designed and used to quantitate these parameters. These models can be combined to efficiently maximize the data from any given subject. RESULTS: Each model leads to a rapid yield of results, with an average turnover of 4.9 days (range 3 to 7 days), and morbidity and mortality were minimal. A combinatorial approach elucidates the precise wound repair deficit of any subject. A case example is presented. CONCLUSIONS: Six surgical models investigating pertinent wound healing parameters are available. A factorial approach of quantitative wound healing assays maximizes data gathered from any one animal, minimizing the number of transgenic and knockout subjects needed; finely dissects molecular pathways of wound healing; and rapidly phenotypes a particular genetically altered mouse. We propose a standardized approach to wound healing assays that will elucidate critical cellular and molecular mechanisms and potential therapies.

Animals↗

PubMed2005

Thymic expression of mutated B16:A preproinsulin messenger RNA does not reverse acceleration of NOD diabetes associated with insulin 2 (thymic expressed insulin) knockout.

We detected insulin2 mRNA but not insulin1 in thymus using real-time PCR analysis. Transgenic expression of a mutated insulin message (alanine rather than tyrosine at insulin B chain amino acid 16) was variably induced in thymus of four transgenic founder strains. The transgenic message levels were as high or higher than native insulin2 message. Lack of the insulin2 gene resulted in the enhancement of anti-insulin autoantibodies (regular NOD vs insulin2-knockout NOD, P<0.001) and in the presence of the B16:A insulin transgenes, levels of insulin autoantibodies remained elevated (regular NOD vs insulin2-knockout NOD with B16:A insulin, P<0.01). Diabetes acceleration by the knockout of the insulin2 gene was not influenced by the presence of the B16:A insulin transgenes. These data suggest that the B16:A insulin does not compensate for lack of native insulin expression in thymus. If lack of thymic insulin message of the insulin2 knockout is the cause of diabetes acceleration, this suggests that native insulin B:9-23 sequences may be crucial in thymus for insulin mediated immunomodulation. Further experiments varying native insulin message expression in thymus is necessary for direct comparison, but the current study provides additional evidence of the potential important role of a specific insulin B chain epitope.

Alanine↗