Construction of a plasmid containing human SMN, the SMA determining gene, coupled to EGFP

We describe here the construction of plasmid pEGFP-C3/SMN, bearing the human SMN gene coupled to the green fluorescent protein (GFP) sequence. The mutation of the SMN gene is responsible for spinal muscular atrophy (SMA), a frequent human infantile genetic disease. We introduced the SMN cDNA into the multiple cloning site of pEGFP-C3. This plasmid bears the neomycin-resistance sequence and the enhanced green fluorescent protein (EGFP). It results in the expression of a fusion protein bearing SMN coupled to a carboxy-terminal GFP tag, used for fluorescence localization studies. Transfection of primary human myoblasts with pEGFP-C3 or pEGFP-C3/SMN revealed that EGFP is intracellularly localized within the cytosol as well as in the nucleus, while the fusion protein EGFP-SMN localized within the nucleus in prominent dot-like structures termed "gems." These data demonstrate that human primary muscle cells can be efficiently transfected and may have important implications for the development of therapeutic strategies in SMA.     

SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens

The spinal muscular atrophy (SMA) region on chromosome 5q13 contains an inverted duplication of about 500 kb, and deleterious mutations in the survival motor neuron 1 (SMN1) gene cause SMA, a common lethal childhood neuropathy. We have used a number of approaches to probe the evolutionary history of these genes and show that SMN gene duplication and the appearance of SMN2 occurred at very distinct evolutionary times. Molecular fossil and molecular clock data suggest that this duplication may have occurred as recently as 3 million years ago in that the position and identity repetitive elements are identical for both human SMN genes and overall sequence divergence ranged from 0.15% to 0.34%. However, these approaches ignore the possibility of sequence homogenization by means of gene conversion. Consequently, we have used quantitative polymerase chain rection and analysis of allelic variants to provide physical evidence for or against SMN gene duplication in the chimpanzee, mankind's closest relative. These studies have revealed that chimpanzees have 2-7 copies of the SMN gene per diploid genome; however, the two nucleotides diagnostic for exons 7-8 and the SMNdelta7 mRNA product of the SMN2 gene are absent in non-human primates. In contrast, the SMN2 gene has been detected in all extant human populations studied to date, including representatives from Europe, the Central African Republic, and the Congo. These data provide conclusive evidence that SMN gene duplication occurred more than 5 million years ago, before the separation of human and chimpanzee lineages, but that SMN2 appears for the first time in Homo sapiens.     

Generation of a tamoxifen inducible SMN mouse for temporal SMN replacement

Proximal spinal muscular atrophy (SMA) is caused by low levels of the SMN protein, encoded by the Survival Motor Neuron genes (SMN1 and SMN2). Mouse models of SMA can be rescued by increased SMN expression, but the timing of SMN replacement for complete rescue is unknown. Studies in zebrafish predict restoration of SMN function during embryogenesis may be important for axonal pathfinding, while the mouse models and normal human disease progression suggest that post-natal treatment may be sufficient for amelioration of disease. To evaluate the timing for SMN replacement, we have generated a stably integrated Cre-inducible SMN mouse in which expression of full-length SMN2 occurs after tamoxifen administration. Our temporally inducible SMN transgene is able to express SMN in embryonic, neonatal, and weanling mice and as such can be utilized in severe and mild SMA mouse models to identify the therapeutic window for SMN replacement.     

Disruption of SMN function by ectopic expression of the human SMN gene in Drosophila

Spinal muscular atrophy is a neurodegenerative disorder caused by mutations or deletions in the survival motor neuron (SMN) gene. We have cloned the Drosophila ortholog of SMN (DmSMN) and disrupted its function by ectopically expressing human SMN. This leads to pupal lethality caused by a dominant-negative effect, whereby human SMN may bind endogenous DmSMN resulting in non-functional DmSMN/human SMN hetero-complexes. Ectopic expression of truncated versions of DmSMN and yeast two-hybrid analysis show that the C-terminus of SMN is necessary and sufficient to replicate this effect. We have therefore generated a system which can be utilized to carry out suppressor and high-throughput screens, and provided in vivo evidence for the importance of SMN oligomerization for SMN function at the level of an organism as a whole.     

Characterisation of novel point mutations in the survival motor neuron gene SMN, in three patients with SMA

We report two novel mutations in three cases of spinal muscular atrophy (SMA), including two distant cousins who followed an unexpectedly severe course. Diagnosis was confirmed by reduced SMN protein and full-length SMN mRNA levels. Sequencing of the non-deleted SMN1 gene revealed a single G insertion at the end of exon 1 in the two cousins and a novel G275S exon 6 missense mutation in the milder case.     

A cell system with targeted disruption of the SMN gene: functional conservation of the SMN protein and dependence of Gemin2 on SMN

The motor neuron degenerative disease spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. Here we report a genetic system developed in the chicken pre-B cell line DT40, in which the endogenous SMN gene is disrupted by homologous recombination, and SMN protein is expressed from a chicken SMN cDNA under control of a tetracycline (tet)-repressible promoter. Addition of tet results in depletion of SMN protein and consequent cell death, which directly demonstrates that SMN is required for cell viability. The tet-induced lethality can be rescued by expression of human SMN, indicating that the function of SMN is highly conserved between the two species. Cells expressing low levels of SMN display slow growth proportional to the amount of SMN they contain. Interestingly, the level of the SMN-interacting protein Gemin2 decreases significantly following depletion of SMN, supporting the conclusion that SMN and Gemin2 form a stable complex in vivo. This system provides a powerful setting for studying the function of SMN in vivo and for screening for potential therapeutics for spinal muscular atrophy.     

人BAFF转基因斑马鱼的构建及早期免疫基因表达检测

目的建立人BAFF转基因斑马鱼模型,探讨其在自身免疫性疾病发病中的作用。方法RT-PCR法由人淋巴瘤细胞克隆了人BAFF基因全长855bp蛋白编码区域,构建表达人BAFF重组质粒Tol2-hBAFF,体外细胞转染并通过免疫印迹法验证蛋白表达。重组载体经显微注射斑马鱼受精卵后,GFP荧光跟踪并筛选阳性鱼。qPCR法检测早期免疫相关基因表达情况。结果人BAFF-GFP融合蛋白可成功表达,利用Tol2-hBAFF重组质粒显微注射斑马鱼受精卵可获得表达人BAFF的转基因斑马鱼,且表达人BAFF斑马鱼1dpf胚胎中TCRAC明显高表达,而Ikaros则表达量显著降低,表明在斑马鱼胚胎中表达人BAFF蛋白会造成早期淋巴系统中基因的过早表达。结论建立的表达人BAFF的转基因斑马鱼,可为系统性红斑狼疮等与BAFF功能亢进密切相关的自身免疫性疾病的治疗,及相关机制研究提供一种具有诸多优点的新型工具。     

Identification of a second presenilin gene in zebrafish with similarity to the human Alzheimer's disease gene presenilin2

Presenilins play prominent roles in the molecular pathogenesis of Alzheimer's disease and during embryo development. We have isolated a zebrafish presenilin orthologue (pre2), which shows a high degree of sequence identity to the human PS2 protein. Zebrafish pre2 is maternally and ubiquitously expressed during early embryo development, whereas Pre2 protein expression is initiated between 6 and 12 hours post fertilisation (hpf), suggesting strict regulation of pre2 translation. pre2 expression is especially high in neural-crest-derived melanocytes.     

Characterization of zebrafish PSD-95 gene family members

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism.     

Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation

Conventional drug discovery approaches require a priori selection of an appropriate molecular target, but it is often not obvious which biological pathways must be targeted to reverse a disease phenotype. Phenotype-based screens offer the potential to identify pathways and potential therapies that influence disease processes. The zebrafish mutation gridlock (grl, affecting the gene hey2) disrupts aortic blood flow in a region and physiological manner akin to aortic coarctation in humans. Here we use a whole-organism, phenotype-based, small-molecule screen to discover a class of compounds that suppress the coarctation phenotype and permit survival to adulthood. These compounds function during the specification and migration of angioblasts. They act to upregulate expression of vascular endothelial growth factor (VEGF), and the activation of the VEGF pathway is sufficient to suppress the gridlock phenotype. Thus, organism-based screens allow the discovery of small molecules that ameliorate complex dysmorphic syndromes even without targeting the affected gene directly.     

Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed,using the Arabidopsis genome sequence and radish genetic mapping

The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by H. C. BeckerS. Giancola and S. Marhadour contributed equally to this work