A recombinant plasmid composed of segments of herpes simplex virus and simian virus 40 viral DNA inserted into the bacterial plasmid pBR322 was microinjected into pronuclei of fertilized mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by the Southern blotting technique for the presence of DNA homologous to the injected plasmid. Two of 78 mice in one series of injections showed clear homology, though the injected sequences had been rearranged. Band intensities from the two positive mice were consistent with the presence of donor DNA in most or all of the cells of the newborns. These results demonstrate that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos. This technique affords the opportunity to study problems of gene regulation and cell differentiation in a mammalian system by application of recombinant DNA technology.

Amyotrophic lateral sclerosis is a progres- (5) to propose that it is a dearth of activity that leads to the neurodegenerative syndrome.Studies of humans with ALS are complicated by the lack of genetic homogeneity or environmental uniformity. Moreover, possible early signs of disease that might appear prior to overt symptoms and could allow preemptive medical intervention are difficult to identify. For these reasons, it is highly desirable to develop an animal model of FALS. The transgenic mouse system (6-8) is well suited to development of such an animal model. Genes introduced into the mouse germ line are often efficiently expressed (9) and can act in a dominant manner to cause diseases that closely resemble the human disorder with respect to phenotype and underlying pathogenetic mechanism (10-12).We have exploited this system to study the role of altered SOD-1 expression in the pathogenesis of ALS and to attempt to develop an animal model of the disease. We introduced a missense mutation, Gly-86 -> Arg, into the fourth exon of a 15-kb mouse genomic clone that we had isolated (13). The same mutation has been observed in the corresponding amino acid residue (position 85) of some patients with FALS (4, 5).In two lines of mice that produced high levels of transgene mRNA in the central nervous system, motor paralysis developed and was associated with degenerative changes of motoneurons within the spinal cord, brain stem, and neocortex. Biochemical measurements of SOD activity in these animals did not reveal a diminution of activity. These findings establish that altered SOD-1 expression can cause motoneuron degeneration and that this degeneration is not associated with reduced SOD activity. Further, these animals constitute a potentially valuable animal model of ALS.

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Genetic material has been successfully transferred into the genomes of newborn mice by injection of that material into pronuclei of fertilized eggs. Initial results indicated two patterns of processing the injected DNA: one in which the material was not integrated into the host genome, and another in which the injected genes became associated with high molecular weight DNA. These patterns are maintained through further development to adulthood. The evidence presented indicates the covalent association of injected DNA with host sequences, and transmission of such linked sequences in a Mendelian distribution to two succeeding generations of progeny.

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The missense mutation Lys-296 --Glu (K296E) in the rhodopsin gene produces an opsin with no chromophore binding site and therefore is not activated by light. Nevertheless, the mutant opsin constitutively activates transducin in vitro and causes photoreceptor degeneration in vivo, possibly by continuously activating the phototransduction cascade, analogous to constant exposure to environmental light. We studied the K296E mutation in eight lines of transgenic mice. Each line developed photoreceptor degeneration with the rate of degeneration increasing monotonically as the ratio of mutant:wild-type opsin mRNA increased. At no time in the course of degeneration was there endogenous light adaptation in the retina as measured by the electroretinogram. The mutant opsin was found to be invariably phosphorylated and stably bound to arrestin. Light-independent activation of transducin was demonstrated only after the removal of arrestin and dephosphorylation of K296E opsin. Thus, K296E opsin in vivo does not activate the phototransduction cascade because it is shut off by photoreceptor inactivation mechanisms. Our data show that the K296E mutation does not cause photoreceptor degeneration by continuous activation of phototransduction.Over 60 different mutations in the rhodopsin gene have been found to cause autosomal dominant retinitis pigmentosa (RP), a progressive degeneration of the neural retina that typically leads to blindness in middle age. The mechanisms by which any of these mutant alleles leads to degeneration of the rod and cone photoreceptors remain obscure. One testable hypothesis (1) is that the mutant opsins continuously activate transducin, resulting in a pathogenic overstimulation of photoreceptors. Support for this hypothesis comes from several observations. Constant exposure to light damages photoreceptor cells (2, 3). Many rhodopsin mutants regenerate poorly with the chromophore, 11-cis-retinal. Opsin, the apoprotein without the chromophore, was reported to activate phototransduction without light [albeit at six orders of magnitude lower efficiency compared to photo-activated metarhodopsin II (4)]. Thus defective regeneration might lead to a pool of weakly active opsin molecules in the photoreceptors. In addition, a rhodopsin mutant, K296E, that causes RP (5, 24) has been found to activate transducin constitutively in vitro (6). To gain insight into the consequences of a constitutively active mutant in vivo, we generated transgenic mice bearing the K296E (Lys-296 -> Glu) mutation. We examined the characteristics of retinal degeneration in these mice and analyzed the mutant opsin from the transgenic mouse retinas. MATERIALS AND METHODSConstruction of Transgene and Generation of Transgenic Mice. The K296E transgene was generated by site-directedThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. mutagenesis on a genomic DNA fragment encompass...

In recent years, several mouse models of amyotrophic lateral sclerosis (ALS) have been developed. One, caused by a G86R mutation in the superoxide dismutase‐1 (SOD‐1) gene associated with familial ALS, has been subjected to extensive quantitative analyses in the spinal cord. However, the human form of ALS includes pathology elsewhere in the nervous system. In the present study, analyses were extended to three motor nuclei in the brainstem. Mutant mice and control littermates were evaluated daily, and mutants, along with their littermate controls, were killed when they were severely affected. Brains were removed after perfusion and processed for Nissl staining, the samples were randomized, and the investigators were blinded to their genetic status. Stereologic methods were used to estimate the number of neurons, mean neuronal volumes, and nuclear volume in three brainstem motor nuclei known to be differetially involved in the human form of the disease, the oculomotor, facial, and hypoglossal nuclei. In the facial nucleus, neuron number consistently declined (48%), an effect that was correlated with disease severity. The nuclear volume of the facial nucleus was smaller in the SOD‐1 mutant mice (45.7% difference from control mice) and correlated significantly with neuron number. The oculomotor and hypoglossal nuclei showed less extreme involvement (<10% neuronal loss overall), with a trend toward fewer neurons in the hypoglossal nucleus of animals with severe facial nucleus involvement. In the oculomotor nucleus, neuronal loss was seen only once in five mice, associated with very severe disease. There was no significant change in the volume of individual neurons in any of these three nuclei in any transgenic mouse. These results suggest that different brainstem motor nuclei are differentially affected in this SOD‐1 mutant model of ALS. The relatively moderate and late involvement of the hypoglossal nucleus indicates that, although the general patterns of neuronal pathology match closely those seen in ALS patients, some differences exist in this transgenic model compared with the progression of the disease in humans. However, these patterns of cellular vulnerability may provide clues for understanding the differential susceptibility of neural structures in ALS and other neurodegenerative diseases. J. Comp. Neurol. 416:112–125, 2000. © 2000 Wiley‐Liss, Inc.

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