Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.

Human major histocompatibility complex class II-restricted T cell responses in transgenic mice.

Transgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying human immunology. However, attempts to obtain human class II-restricted T cell responses in such transgenic mice have had only limited success, possibly due to an inability of mouse CD4 to interact efficiently with human MHC class II molecules. To circumvent this problem, we constructed recombinant MHC class II genes in which the peptide-binding domain was derived from human DR sequences whereas the CD4-binding domain was derived from mouse I-E sequences. Purified chimeric human/mouse MHC class II molecules were capable of specifically binding DR-restricted peptides. Human B cell transformants that expressed these chimeric MHC class II molecules could present peptide antigens to human T cell clones. Expression of these chimeric class II molecules in transgenic mice led to the intrathymic deletion of T cells expressing superantigen-reactive V beta gene segments, indicating that the chimeric class II molecules could influence the selection of the mouse T cell repertoire. These transgenic mice were fully capable of mounting human DR-restricted immune responses after challenge with peptide or whole protein antigens. Thus, the chimeric class II molecules can serve as functional antigen presentation molecules in vivo. In addition, transgenic mice expressing chimeric class II molecules could be used to generate antigen-specific mouse T cell hybridomas that were capable of interacting with human antigen-presenting cells.

Mouse oocytes transcribe injected Xenopus 5S RNA gene.

Transcripts produced after injection of the Xenopus 5S RNA gene into oocyte germinal vesicles of mice migrate electrophoretically with the 5S RNA marker, an indication that the gene is transcribed and processed with considerable accuracy. Approximately two 5S RNA molecules are transcribed per gene per hour. This system may be useful in studying DNA processing and gene regulation by the mammalian ovum and might be modified to allow permanent incorporation of specific genes into mice.

Mutant human presenilin 1 protects presenilin 1 null mouse against embryonic lethality and elevates Abeta1-42/43 expression.

Mutations in presenilin 1 (PS1) are linked to early onset of familial Alzheimer's disease (FAD) and are shown to foster production of Abeta1-42/43 in FAD patients and transgenic mice. PS1 null mice are embryonic lethal and exhibit axial skeleton malformation and CNS defects. We show that transgenic mouse lines expressing either the wild-type human PS1 protein or human PS1 with the A246E FAD mutation can rescue the PS1 knockout mouse from embryonic lethality to similar degrees, indicating that the mutation does not lead to loss of PS1 function during development. Furthermore, a 50% reduction of PS1 activity in PS1(+/-) mice does not lead to Abeta1-42/43 increase, whereas expression of human mutant PS1 on murine PS1 null background is sufficient to elevate Abeta1-42/43, supporting a gain-of-function activity as the result of the PS1 mutation.

Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor.

For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.

Somatostatin receptor subtype 2 knockout mice are refractory to growth hormone-negative feedback on arcuate neurons.

The pulsatile nature of GH release is apparently regulated by alternating sequential changes in two hypothalamic hormones, GH releasing hormone (GHRH) and somatostatin. Entrainment of this pulsatility appears to involve GH-mediated negative feedback. Recently a new receptor involved in GH release was cloned. Activation of this receptor by GH-releasing peptides and MK-0677 initiates and amplifies GH pulsatility and is associated with increased Fos immunoreactivity and electrical activity in GHRH containing arcuate neurons. We show that pretreating mice with GH blocks activation of these neurons by MK-0677. Similarly, octreotide inhibited the action of MK-0677. To determine whether this GH-mediated negative feedback on GHRH neurons was direct, or by GH stimulation of somatostatin release from periventricular neurons, we selectively inactivated the gene for one of the five specific somatostatin receptor subtypes (subtype 2). In the knockout mice, both GH and octreotide failed to inhibit MK-0677 activation of arcuate neurons. GH did, however, increase Fos immunoreactivity in the periventricular nucleus, consistent with GH stimulation of somatostatin release from periventricular neurons. Thus, GH-mediated negative feedback involves signaling between periventricular and arcuate neurons with the signal being transduced specifically through somatostatin subtype 2 receptors.

Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein.

Ghrelin, a stomach-derived orexigenic hormone, has stimulated great interest as a potential target for obesity control. Pharmacological evidence indicates that ghrelin's effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. These include intracerebroventricular application of antibodies to neutralize NPY and AgRP, and the application of an NPY Y1 receptor antagonist, which blocks some of the orexigenic effects of ghrelin. Here we describe treatment of Agrp(-/-);Npy(-/-) and Mc3r(-/-);Mc4r(-/-) double knockout mice as well as Npy(-/-) and Agrp(-/-) single knockout mice with either ghrelin or an orally active nonpeptide ghrelin agonist. The data demonstrate that NPY and AgRP are required for the orexigenic effects of ghrelin, as well as the involvement of the melanocortin pathway in ghrelin signaling. Our results outline a functional interaction between the NPY and AgRP pathways. Although deletion of either NPY or AgRP caused only a modest or nondetectable effect, ablation of both ligands completely abolished the orexigenic action of ghrelin. Our results establish an in vivo orexigenic function for NPY and AgRP, mediating the effect of ghrelin.

Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice.

Amyloid precursor protein (APP) is a member of a larger gene family including amyloid precursor-like proteins (APLP), APLP2 and APLP1. To examine the function of APLP2 in vivo, we generated APLP2 knockout (KO) mice. They are of normal size, fertile, and appear healthy up to 22 months of age. We observed no impaired axonal outgrowth of olfactory sensory neurons following bulbectomy, suggesting against an important role for APLP2 alone in this process. Because APLP2 and APP are highly homologous and may serve similar functions in vivo, we generated mice with targeted APLP2 and APP alleles. Approximately 80% of double KO mice die within the first week after birth, suggesting that APLP2 and APP are required for early postnatal development. The surviving approximately 20% of double KO mice are 20-30% reduced in weight and show difficulty in righting, ataxia, spinning behavior, and a head tilt, suggesting a deficit in balance and/or strength. Adult double KO mice mate poorly, despite apparent normal ovarian and testicular development. Otherwise, double KO mice appear healthy up to 13 months of age. We conclude, that APLP2 and APP can substitute for each other functionally.

Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the beta-amyloid precursor protein.

Mutations in the beta-amyloid precursor protein are strongly associated with some cases of familial Alzheimer's disease. The normal physiological role of beta-amyloid precursor protein in the brain was evaluated in a cross-sectional analysis of mice deficient in beta-amyloid precursor protein. Compared with wild-type control mice the beta-amyloid precursor protein-null mice developed age-dependent deficits in cognitive function and also had impairments in long-term potentiation. In addition, the brains of the beta-amyloid precursor protein-null mice had marked reactive gliosis in many areas, especially in the cortex and hippocampus. A subpopulation of mice (n = 15) died prematurely (between three and 18 months of age). Analysis of another six mice from the same population that were showing weight loss and hypolocomotor activity exhibited a marked reactive gliosis as detected by immunoreactivity for glial fibrillary acidic protein and a profound loss of immunoreactivities for the presynaptic terminal vesicle marker proteins synaptophysin and synapsin and the dendritic marker microtubule-associated protein-2 in many brain areas, but most predominantly in the cortex and hippocampus. These results suggest that normal beta-amyloid precursor protein may serve an essential role in the maintenance of synaptic function during ageing. A compromise of this function of the beta-amyloid precursor protein may contribute to the progression of the memory decline and the neurodegenerative changes seen in Alzheimer's disease.

Mice deficient for the amyloid precursor protein gene.

To understand the in vivo function of the amyloid precursor protein (APP) we generated an APP null mutation in mice by homologous recombination in embryonic stem (ES) cells. We show here that homozygous APP deficient mice were produced at expected frequencies. Neither APP mRNA nor protein could be detected in these animals. Yet the homozygous APP mutant mice are fertile and do not show overt abnormalities at up to 12 weeks of age. Neuroanatomical studies of the brain did not reveal significant differences in the knockout mice as compared to the wild-type controls. These results argue against an essential function of APP in mouse embryonic and early neuronal development.

Induction of neuropeptide Y expression in dorsomedial hypothalamus of diet-induced obese mice.

Alterations of hypothalamic neuropeptide Y(NPY) and melanocortinergic functions in diet-induced obese (DIO) C57BL/6J mice were investigated by in situ hybridization. Compared with controls, the DIO mice displayed a profound induction (approximately 40-fold) of NPY expression in the dorsomedial (DMH) and ventromedial (VMH) hypothalamic nuclei, whereas the level of NPY mRNA in the arcuate nucleus (ARC) was reduced by 44%. The expression of pro-opiomelanocortin (POMC) and agouti-related protein was not significantly altered in the ARC of obese mice. Both excess body weight gain and altered hypothalamic NPY expression were reversible. We propose that the highly induced NPY expression in DMH and/or VMH may be a contributing etiological factor for the development of obesity and leptin resistance in the DIO mice.

Induction of c-fos mRNA in the arcuate nucleus of normal and mutant growth hormone-deficient mice by a synthetic non-peptidyl growth hormone secretagogue.

We have studied by in situ hybridization histochemistry the mRNA expression of the c-fos immediate early gene in the brains of wild type and dwarf(dw/dw) and little(lit/lit) mutant mice after systemic injections of the synthetic GH secretagogues GHRP-6 and L-163,191. Both GH secretagogues induced a marked c-fos mRNA expression in the arcuate-ventromedial hypothalamus (ARC-VMH) of both control and mutant mice indicating a possible action on growth hormone releasing hormone (GHRH) neurones in the ARC-VMH. Both dw/dw and lit/lit mice showed a 5-fold elevation in GHRH mRNA expression in the ARC-VMH compared with control animals under basal conditions. Since lit/lit mice have a reduced ability to secrete GH and lack a functional GHRH receptor while dw/dw mice lack both GH and presumably GHRH receptors, the GH-secretagogue-induced c-fos mRNA in the brain of these mutants are unlikely to be mediated by an indirect action of GH or a interaction of the synthetic GH-secretagogue with the GHRH receptor.

Differential regulation of neuropeptide Y receptors in the brains of NPY knock-out mice.

To study the effect of NPY deletion on the regulation of its receptors in the NPY knockout (NPY KO) mice, the expression and binding of NPY receptors were investigated by in situ hybridization and receptor autoradiography using (125)I-[Leu(31),Pro(34)]PYY and (125)I-PYY(3-36) as radioligands. A 6-fold increase in Y2 receptor mRNA was observed in the CA1 region of the hippocampus in NPY KO mice, but a significant change could not be detected for Y1, Y4, Y5 and y6 receptors. Receptor binding reveals a 60-400% increase of Y2 receptor binding in multiple brain areas. A similar increase in Y1 receptor binding was seen only in the hypothalamus. These results demonstrate the NPY receptor expression is altered in mice deficient for its natural ligand.

Effect of target gene CpG content on spontaneous mutation in299 transgenic mice.

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct transgenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10-5 range and were predominantly base pair substitutions, similar to results seen in Big Blue. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC-->AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.

Translation of globin messenger RNA by the mouse ovum.

It has been demonstrated that the Xenopus oocyte can translate rabbit haemoglobin messenger RNA (mRNA) following microinjection of the message into the cell. The Xenopus oocyte has since been shown to be capable of translating a variety of messenger RNAs from different species. This system has proved useful in un-erstanding the mechanism of message translation and has also provided information about the translation capability of the Xenopus oocyte. Several other cell types, including HeLa cells and fibroblasts, can also translate exogenous message injected into the cell. However, there have been no reports of injection of mRNA into oocytes or fertilised one-cell ova of mammalian species. Nevertheless, the latter system could be of considerable use in studying the processing of exogenous messages in a mammalian system undergoing development, as well as providing insight into the way the early embryo processes injected messages and the protein products of such messages. We report here the results of injecting message into the fertilised one-cell mouse ovum and show that both mouse and rabbit globin mRNA are translated in this system.

Generation of mice expressing the human glucagon receptor with a direct replacement vector.

The process of evaluating the in vivo efficacy of non-peptidyl receptor antagonists in animal models is frequently complicated by failure of compounds displaying high affinity against the human receptors to show measurable affinity at the corresponding rodent receptors. In order to generate a suitable animal model in which to evaluate the in vivo activity of non-peptidyl glucagon receptor antagonists, we have utilized a direct targeting approach to replace the murine glucagon receptor with the human glucagon receptor gene by homologous recombination. Specific expression of the human glucagon receptor (GR) in the livers of transgenic mice was confirmed with an RNase protection assay, and the pharmacology of the human GRs expressed in the livers of these mice parallels that of human GR in a recombinant CHO cell line with respect to both binding of 125I-glucagon and the ability of glucagon to stimulate cAMP production. L-168,049, a non-peptidyl GR antagonist selective for the human GR shows a 3.5 fold higher affinity for liver membrane preparations of human GR expressing mice (IC50 = 172 +/- 98 nM) in the presence of MgCl2 in marked contrast to the measured affinity of the murine receptor (IC50 = 611 +/- 197 nM) for this non-peptidyl antagonist. The human receptors expressed are functional as measured by the ability of glucagon to stimulate cAMP production and the selectivity of this antagonist for the human receptor is further verified by its ability to block glucagon-stimulated cyclase activity with 5 fold higher potency (IC50 = 97.2 +/- 13.9 nM) than for the murine receptor (IC50 = 504 +/- 247 nM). Thus we have developed a novel animal model for evaluating GR antagonists in vivo. These mice offer the advantage that the regulatory sequences which direct tissue specific and temporal expression of the GR have been unaltered and thus expression of the human gene in these mice remains in the normal chromosomal context.

Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs.

A plasmid, designated pMK, containing the structural gene for thymidine kinase from herpes simplex virus (HSV) fused to the promoter/regulatory region of the mouse metallothionein-I gene, was injected into the pronucleus of fertilized one-cell mouse eggs; the eggs were subsequently reimplanted into the oviducts of pseudopregnant mice. The first experiment produced 19 offspring, one of which expressed high levels of HSV thymidine kinase activity in the liver and kidney. pMK DNA sequences were detected in equal amounts in several tissues of the expressing mouse as well as in three mice that did not express HSV thymidine kinase activity. In all cases, several copies of the pMK plasmid were tandemly duplicated and integrated into mouse DNA. It appears as though multiple copies of the intact plasmid were fused by homologous recombination either before or after integration at a single site in the mouse genome. The overall efficiency of obtaining somatic expression of thymidine kinase in experiments performed to date is about 10% (4/41), and twice this number have integrated pMK DNA. This procedure not only provides a means of introducing new genes into mice, but it will also be a valuable system for studying tissue-specific regulation of gene expression.

Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.

Microinjection of foreign DNA into fertilized mammalian eggs is a convenient means of introducing genes into the germ line. Some of the more important parameters that influence successful integration of foreign DNA into mouse chromosomes are described. The effects of DNA concentration, size, and form (supercoiled vs. linear with a variety of different ends) are considered as well as the site of injection (male pronucleus, female pronucleus, or cytoplasm) and buffer composition. The optimal conditions for integration entail injection of a few hundred linear molecules into the male pronucleus of fertilized one-cell eggs. Under these conditions about 25% of the mice that develop inherit one or more copies of the microinjected DNA. The overall efficiency also depends on the choice of mouse strains; for example, generating transgenic mice that express foreign growth hormone genes is about eight times easier with C57/BL6 X SJL hybrid mice than with inbred C57/BL6 mice.

Resistance to fever induction and impaired acute-phase response in interleukin-1 beta-deficient mice.

We used gene targeting in embryonic stem cells to introduce an IL-1 beta null allele in mice. The IL-1 beta-deficient mice develop normally and are apparently healthy and fertile. The IL-1 beta null mice responded normally in models of contact and delayed-type hypersensitivity or following bacterial endotoxin LPS-induced inflammation. The IL-1 beta-deficient mice showed equivalent resistance to Listeria monocytogenes compared with wild-type controls. In contrast, when challenged with turpentine, which causes localized inflammation and tissue injury, the IL-1 beta mutant mice exhibited an impaired acute-phase inflammatory response and were completely resistant to fever development and anorexia. These results highlight a central role for IL-1 beta as a pyrogen and a mediator of the acute-phase response in a subset of inflammatory disease models, and support the notion that blocking the action of a single key cytokine can alter the course of specific immune and inflammatory responses.

Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.

Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.