Targeted expression of a toxin gene to D1 dopamine receptor neurons by cre-mediated site-specific recombination.

Idiopathic Parkinson's disease involves the loss of midbrain dopaminergic neurons, resulting in the presynaptic breakdown of dopaminergic transmission in the striatum. Huntington's disease and some neurodegenerative diseases with Parkinsonian features have postsynaptic defects caused by striatal cell death. Mice were generated in which an attenuated form of the diphtheria toxin gene (tox-176) was expressed exclusively in D1 dopamine receptor (D1R)-positive cells with the aim of determining the effect of this mutation on development of the basal ganglia and on the locomotor phenotype. Transgenic mice expressing Cre, a site-specific DNA recombinase, were crossed with a second line in which a transcriptionally silenced tox-176 gene was inserted into the D1R gene locus by homologous recombination. Young doubly transgenic mutant mice expressing the tox-176 gene displayed bradykinesia, dystonia, and had falls caused by myoclonic jerks. The mutant brain had evidence of apoptosis and reactive gliosis and, consistent with the D1R expression pattern, the striatum was reduced in volume, and the Islands of Calleja were absent. In contrast, the cortex was of normal thickness. D1Rs were not detectable in mutants by in situ hybridization or ligand autoradiography, whereas D2 dopamine receptor (D2R) mRNA and protein was present in the striatum. In addition, substance P and dynorphin, neuropeptides known to be expressed in D1R-positive striatonigral projection neurons were not detectable. Enkephalin, a marker found in D2-positive striatopallidal projection neurons was expressed in the mutant brain. The mutant represents a novel neurodegenerative disease model with a dramatic extrapyramidal phenotype.

Microinjection of in vitro transcribed RNA and antisense oligonucleotides in mouse oocytes and early embryos to study the gain- and loss-of-function of genes.

Mouse oocytes have proven useful in experiments aimed at studying gene function. They have been used to analyze the gain-of-function acquired after microinjection of RNA transcribed in vitro from specific gene constructs, and for establishing loss-of-function mutation obtained by injecting in vitro transcribed antisense RNA and/or synthetic oligonucleotides. This article presents protocols utilized in these studies. Specifically, the acquisition of mouse oocytes and/or embryos, the genesis of the necessary DNA and/or RNA to be used, and procedures for microinjection.

Down's syndrome-like skeletal abnormalities in Ets2 transgenic mice.

Expression of Ets2, a proto-oncogene and transcription factor, occurs in a variety of cell types. During murine development it is highly expressed in newly forming cartilage, including in the skull precursor cells and vertebral primordia. Ets2 is located on human chromosome 21 (ref. 8) and is overexpressed in Down's syndrome (trisomy 21). Here we generate transgenic mice to investigate the consequences of overexpression of Ets2. We find that mice with less than 2-fold Ets2 overexpression in particular organs develop neurocranial, viscerocranial and cervical skeletal abnormalities. These abnormalities have similarities with the skeletal anomalies found in trisomy-16 mice and humans with Down's syndrome, in which the gene dosage of Ets2 is increased. Our results indicate that Ets2 has a role in skeletal development and implicate the overexpression of Ets2 in the genesis of some skeletal abnormalities that occur in Down's syndrome.

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.