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  • The 16th International Mouse Genome Conference (2002)
    of Genomes Attendees Sponsors Table of Contents Photographs Awards POSTER 76 GENE ENTRAPMENT STRATEGY FOR PROTEOMICS A Osipovich Vanderbilt University Ruley E Vanderbilt University To facilitate genetic and biochemical analysis of mammalian gene functions we have developed a gene entrapment strategy that uses a new gene trap vector LNPAT1 The vector functions as a poly A trap targeting most genes regardless whether they are expressed in ES cells Disrupted genes are identified by sequencing of 3 RACE products which are used for preparation of DNA microarray chips providing a resource to study expression profiles of genes in the mutant library The LNPAT1 vector incorporates an enhanced green fluorescent protein EGFP reporter to monitor gene expression in vitro and in tissues of an intact animal The vector also contains heterotypic loxP sites for Cre mediated cassette replacement allowing integrated vectors and genes trapped in ES cells to be engineered for a variety of applications e g a to optimise poly A gene trap vectors b to reverse null mutations c to create heteromorphic alleles of disrupted genes d for chromosome engineering e to affinity tag cellular proteins to characteriseinteracting proteins from normal tissues of the mouse Depending on what part of

    Original URL path: http://www.imgs.org/Archive/abstracts/2002abstracts/file76.shtml (2016-02-17)
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  • The 16th International Mouse Genome Conference (2002)
    W 1 Ball S 2 Plagge A 1 Maconochie M 2 Kelsey G 1 Williamson C 1 MRC Mammalian Genetics Unit 2 The Babraham Institute An increasing number of imprinted genes have antisense RNAs associated with them and some have been shown to play an active role in genomic imprinting Within the Gnas complex in distal mouse chromosome 2 are several antisense RNAs all of which are imprinted paternally expressed and non coding In addition to an unspliced form of about 30 kb there are a number of alternatively spliced forms that are up to 1 4 kb in length These antisense RNAs are called Nespas Nespas overlaps the maternally expressed gene Nesp within the Gnas complex but is unlikely to regulate the expression of Nesp in mid gestation embryos because Nespas and Nesp are expressed in different sites Nespas may regulate Nesp at earlier developmental stages Other studies to examine the role of Nespas have been hampered by the close proximity of Gnasxl a paternally expressed protein encoding gene Analysis of the 2 kb of sequence that separates the first exons of Nespas and Gnasxl has shown that their minimal promoter regions do not overlap and that the Nespas

    Original URL path: http://www.imgs.org/Archive/abstracts/2002abstracts/file77.shtml (2016-02-17)
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  • The 16th International Mouse Genome Conference (2002)
    on Aging NIH Martin P Bassey U Stagg C 1 Ko M NIA In order to recover long insert cDNA clones for genes expressed only at limited times and places during embryogenesis we have developed a method to construct a cDNA library from small numbers of cells 600 800cells The method allows us to generate cDNA libraries enriched for long transcripts without size selection of insert DNAs After cDNA synthesis

    Original URL path: http://www.imgs.org/Archive/abstracts/2002abstracts/file78.shtml (2016-02-17)
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  • The 16th International Mouse Genome Conference (2002)
    S Temple University School of Medicine Previous studies have suggested that disparate mutant alleles of the t complex localized testis specific axonemal dynein heavy chain axDHC gene Dnahc8 are responsible for two very different male sterility phenotypes a sperm flagellar motility defect and a flagellar morphogenesis deficiency respectively Initial cloning of full length Dnahc8 cDNAs revealed two transcripts coding for proteins with different C termini and a non conserved N terminal extension replete with effector binding ligands and nearby O phosphorylatable O GlcNacylatable residues suggesting possible Yin Yang regulation of ligand binding activity However northern analyses have suggested that other Dnahc8 5 ends exist encoding proteins lacking this N terminal extension To further explore this possibility experiments to isolate and characterize all possible and t mRNA 5 ends are currently underway Our initial evidence indicates that 5 ends coding for shortened N termini probably do not exist however numerous 5 UTR cDNA clones show internal deletions or small additions while all are identical for their final 45 or 34 bases and t ends respectively Sequencing across the genomic region containing the 5 UTRs shows that this 45 34 base region is at the 5 end of the 2nd exon 10

    Original URL path: http://www.imgs.org/Archive/abstracts/2002abstracts/file79.shtml (2016-02-17)
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  • The 16th International Mouse Genome Conference (2002)
    Temple University School Of Medicine The Hst6 locus maps to In 17 4 of the mouse t complex and contains one or more genes that cause male sterility via deleterious effects on sperm flagellar movement and ultrastructure While a prime candidate for both effects is an axonemal dynein heavy chain gene Dnahc8 the genomic region immediately flanking the Dnahc8 locus contains two additional testis specifically expressed genes TsgL1 and Tsga2 whose expression characteristics differ significantly between t haplotype t and wildtype alleles Tsga2 located just distal to Dnahc8 is a rather unremarkable gene producing an abundant transcript of 1 kb with a 906 bp ORF Expression is reduced in the testes of t homozygotes and TSGA2t contains 14 amino acid changes most within the N terminal two thirds of the ORF relative to TSGA2 In situ hybridization studies indicate that Tsga2 expression is abundant in pachytene spermatocytes with progressively decreasing levels in round and elongating spermatids consistent with the previously reported localization of TSGA2 in late spermatocytes and round spermatids TsgL1 located just proximal to Dnahc8 shows complex organizational and expression patterns Multiple messages are transcribed from this 350 kb locus the three smallest 3 5 2 5 and 1

    Original URL path: http://www.imgs.org/Archive/abstracts/2002abstracts/file80.shtml (2016-02-17)
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  • The 16th International Mouse Genome Conference (2002)
    Nebraska Elo KT Nielsen MK Pomp D Department of Animal Science University of Nebraska In the mouse many genes with known physiological relevance to energy regulating pathways have been discovered while several predisposition genes QTL are being identified Given that few relevant mutations have been identified within these physiological genes that explain variation in segregating populations it is interesting to speculatively hypothesize that predisposition and physiological genes represent different loci To test this theory we are conducting experiments to identify QTL regulating the expression of genes with physiological relevance to energy balance In the present study we conducted a QTL scan using an F2 population n 636 derived from two mouse lines divergently selected for heat loss The inbred high IH heat loss mice have 50 greater heat loss 35 less body fat 20 greater feed intake and two fold greater activity levels compared to the inbred low IL heat loss mice Yet both lines are similar in body weight Previously we have shown differential mRNA expression of hypothalamic oxytocin Oxt tissue inhibitor of metalloproteinase 2 Timp 2 and ribosomal protein L3 Rpl3 between the IH and IL lines Evidence for QTL controlling Oxt and Rpl3 expression were detected on

    Original URL path: http://www.imgs.org/Archive/abstracts/2002abstracts/file81.shtml (2016-02-17)
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  • The 16th International Mouse Genome Conference (2002)
    Poirier C 1 2 Bishop CE 1 Department of Obstetrics Gynecology 2 Department of Molecular and Human Genetics Baylor College of Medicine The recessive non pleiotropic mouse mutation jsd juvenile spermatogonial depletion is characterized by sterility in adult males The testes of young males homozygous for the mutation undergo a single wave of spermatogenesis after which type A spermatogonial stem cells fail to differentiate Using stem cell transplantation we have previously shown that the defect is cell autonomous being restricted to the germ line rather than the supporting tissue We have localized jsd to an interval of approximately 0 4cM at 49cM on chromosome 1 between markers D1Mit215 and 257SP6 a distance of approximately 1 5Mb At least 8 known or novel genes have been identified in this interval We have chosen one gene termed Jr1 as a primary candidate for jsd based on a number of criteria including its restricted spatio temporal expression pattern in the male germ line as revealed by in situ hybridization The JR1 protein contains a single FYVE domain suggesting that it localizes to endosomes and multiple WD40 domains putatively involved in protein protein interactions Jr1 is well conserved and its human homologue FENS1 has

    Original URL path: http://www.imgs.org/Archive/abstracts/2002abstracts/file82.shtml (2016-02-17)
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  • The 16th International Mouse Genome Conference (2002)
    Institute of Psychiatry The arrays of in situ synthesized oligonucleotides produced by Affymetrix currently offer the best standardised method of large scale measurement of transcript levels Data can be compared relatively well between experiments including those in different laboratories The oligonucleotides probes are 25 bases long and for each a single mismatch probe is used as a specificity control One recurring problem with processing data from these chips is that many mismatch probes give a higher signal intensity than their perfect match counterparts Several methods have been proposed for background removal and normalisation but none so far use of prediction of duplex affinity from probe sequences A duplex melting temperature Tm criterion is used by Affymetrix in their proprietary probe selection method but not in analysis of hybridisation data A reasonable prediction of binding affinities can be made using the nearest neighbour model although some of the probes may not show simple two state melting Applying this method to the Affymetrix U74Av2 probe list gives a Tm range of over 40º Most hybridisations show a modest effect of predicted Tm on signal intensity The fact that the effect is modest indicates that the methods have been successfully optimised to not

    Original URL path: http://www.imgs.org/Archive/abstracts/2002abstracts/file83.shtml (2016-02-17)
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