Rapid Construction in Yeast of Complex Targeting Vectors for Gene Manipulation in the Mouse

Storck, Thorsten; Krüth, Ulrich; Kolhekar, R.; Sprengel, Rolf; Seeburg, Peter H.

doi:10.1093/nar/24.22.4594

Cited by 47 publications

(31 citation statements)

References 10 publications

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“…The targeting vector was the YCplac22 shuttle vector containing a 21-kb NotI-SalI fragment of a murine gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67), spanning parts of exon 1 up to intron 6 (about 19 kb downstream of the start codon) (11). The dicistronic GluR-B-IRES-lacZ cassette was introduced 30 bp upstream of the translational start codon of the GAD67 gene by using homologous recombination in yeast (12). The rat glutamate receptor B subunit (GluR-B) cDNA encoding the Q͞R site edited in the flip configuration was used, and it contains 200 bp of the 5Ј untranslated region (13).…”

Section: Methodsmentioning

confidence: 99%

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

Fuchs

Doheny

Faulkner

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

116

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Gamma oscillations synchronized between distant neuronal populations may be critical for binding together brain regions devoted to common processing tasks. Network modeling predicts that such synchrony depends in part on the fast time course of excitatory postsynaptic potentials (EPSPs) in interneurons, and that even moderate slowing of this time course will disrupt synchrony. We generated mice with slowed interneuron EPSPs by gene targeting, in which the gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was altered to drive expression of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit GluR-B. GluR-B is a determinant of the relatively slow EPSPs in excitatory neurons and is normally expressed at low levels in ␥-aminobutyric acid (GABA)ergic interneurons, but at high levels in the GAD-GluR-B mice. In both wild-type and GAD-GluR-B mice, tetanic stimuli evoked gamma oscillations that were indistinguishable in local field potential recordings. Remarkably, however, oscillation synchrony between spatially separated sites was severely disrupted in the mutant, in association with changes in interneuron firing patterns. The congruence between mouse and model suggests that the rapid time course of AMPA receptor-mediated EPSPs in interneurons might serve to allow gamma oscillations to synchronize over distance.G amma-frequency (30-to 90-Hz) neuronal oscillations, occurring in a non-time-locked fashion in response to visual stimuli, have been proposed to encode global features of spatially extended stimuli (1). A crucial physiological question concerns how synchrony arises, given the expected slow axonal conduction times in the cortex, estimated to be as low as 0.10-0.15 mm͞ms (2, 3).Network simulations (containing model pyramidal cells, interneurons, and axon conduction delays) predict that tight synchrony between distant sites could arise as an emergent property-without a central pacemaker-when interneurons fire in doublets (4-7). Precise timing of the doublet intervaldetermined in part by ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor mediated excitation from pyramidal cells, especially cells at a distance from the postsynaptic interneuron-provides a signal encoding phase lags between separated neurons; this signal would provide feedback to nearby pyramidal cells, in the form of synaptic inhibition, which would tend to correct for phase differences (ref. 8; Appendix, which is published as supplemental data on the PNAS web site, www.pnas.org).Here we use a combination of simulation, transgenic, and electrophysiological techniques to show that interneuron doublets are necessary for two-site synchrony to occur and that they critically depend on excitatory postsynaptic potential (EPSP) kinetics in interneurons. Experimental ProceduresSimulations. Network simulations of gamma oscillations were performed by using the program described in Traub et al. (5).Briefly, the network consisted of a 96 ϫ 32 cell array of pyramidal neurons, and a superimp...

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Section: Methodsmentioning

confidence: 99%

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

Fuchs

Doheny

Faulkner

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

116

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“…This 7.9-kb fragment was further subcloned, sequenced, and found to contain exons 21-28 of the mouse Fancd2 gene. The following primer pairs were used to amplify sequence flanking exons 26 and 27, and the PCR products were cloned into the plasmid pRAY-1 (Storck et al 1996) to generate pRAY-1+ flanking arms: 5Ј-TCAGCCTCACATGGA GTTTAACG-3Ј/5Ј-GTGTGGACACTAACCTCACTCGC-3Ј and 5Ј -TTTCCTGTCCTCATCTGCG-3Ј/5Ј-TGCAAAGAGAGAAA TGACTCAAG-3Ј. pRAY-1+ flanking arms and the YCplac22-TK + BamHI fragment were cotransfected into yeast strain MW3317-21A (−trp, −ura) and selected in −ura/−trp dropout media.…”

Section: Targeting Vector Design and Generation Of Fancd2-deficient Micementioning

confidence: 99%

Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice

Houghtaling¹,

Timmers²,

Noll³

et al. 2003

Genes Dev.

272

298

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Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA damage, bone marrow failure, congenital defects, and cancer. To further investigate the in vivo function of the FA pathway, mice with a targeted deletion in the distally acting FA gene Fancd2 were created. Similar to human FA patients and other FA mouse models, Fancd2 mutant mice exhibited cellular sensitivity to DNA interstrand cross-links and germ cell loss. In addition, chromosome mispairing was seen in male meiosis. However, Fancd2 mutant mice also displayed phenotypes not observed in other mice with disruptions of proximal FA genes. These include microphthalmia, perinatal lethality, and epithelial cancers, similar to mice with Brca2/Fancd1 hypomorphic mutations. These additional phenotypes were not caused by defects in the ATM-mediated S-phase checkpoint, which was intact in primary Fancd2 mutant fibroblasts. The phenotypic overlap between Fancd2-null and Brca2/Fancd1 hypomorphic mice is consistent with a common function for both proteins in the same pathway, regulating genomic stability.[Keywords: Fanconi anemia; cancer; Fancd2; Brca2; DNA repair; chromosome pairing] Supplemental material is available at http://www.genesdev.org.

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“…Next, we modified a second plasmid (pRay-1) containing a cassette comprising the neomycin resistance gene under the control of the thymidine kinase promoter, the yeast selection marker URA3, and the lacZ reporter gene in front of these selection markers by inserting 400-bp recombinogenic arms on either side of the cassette. These arms were generated by PCR from the mouse DNA clone and were designed to allow homologous recombination in yeast so as to insert the lacZ gene in frame with the first ATG of FAT1, hence generating a targeting vector (43).…”

Section: Generation Of Fat-deficient Micementioning

confidence: 99%

Mice Lacking the Giant Protocadherin mFAT1 Exhibit Renal Slit Junction Abnormalities and a Partially Penetrant Cyclopia and Anophthalmia Phenotype

Ciani

Patel

Allen

et al. 2003

Molecular and Cellular Biology

219

184

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While roles in adhesion and morphogenesis have been documented for classical cadherins, the nonclassical cadherins are much less well understood. Here we have examined the functions of the giant protocadherin FAT by generating a transgenic mouse lacking mFAT1. These mice exhibit perinatal lethality, most probably caused by loss of the renal glomerular slit junctions and fusion of glomerular epithelial cell processes (podocytes). In addition, some mFAT1 ؊/؊ mice show defects in forebrain development (holoprosencephaly) and failure of eye development (anophthalmia). In contrast to Drosophila, where FAT acts as a tumor suppressor gene, we found no evidence for abnormalities of proliferation in two tissues (skin and central nervous system [CNS]) containing stem and precursor cell populations and in which FAT is expressed strongly. Our results confirm a necessary role for FAT1 in the modified adhesion junctions of the renal glomerular epithelial cell and reveal hitherto unsuspected roles for FAT1 in CNS development.Cell adhesion molecules are essential for the regulation of coordinated changes in cell shape and position that occur during morphogenesis and also for the maintenance of cell-cell interactions once development is complete. The best-studied families of cell adhesion molecules are the cadherins. These were originally identified as Ca 2ϩ -dependent cell-cell adhesion proteins characterized by five repeated cadherin-specific motifs in their extracellular domain. This domain is an approximately 110-amino-acid peptide that mediates homophilic interactions with other cadherin molecules, forming dimers which then interact with dimers on neighboring cells (41,45,46). The cytoplasmic domain of these so-called classical fiverepeat cadherins is well conserved between different members of the family, containing motifs required for interaction with catenins that can then mediate linkage to the cytoskeleton (13,17,34,42). In addition to the classical cadherins, at least five other classes of cadherins have been identified: nonclassical cadherins, in which the first extracellular repeat does not contain the HAV (histidine-alanine-valine) cell adhesion sequence present in all classical cadherins, and four additional families. Two of these, the desmosomal adhesion molecules desmocollins and desmogleins, all share the five extracellular cadherin repeat structure with the sole exception of desmocollin-1. Two other families, the flamingo cadherins and protocadherins, have variable numbers of cadherin repeats and additional motifs as well as differing cytoplasmic domain sequences (30,44).

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Rapid Construction in Yeast of Complex Targeting Vectors for Gene Manipulation in the Mouse

Cited by 47 publications

References 10 publications

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice

Mice Lacking the Giant Protocadherin mFAT1 Exhibit Renal Slit Junction Abnormalities and a Partially Penetrant Cyclopia and Anophthalmia Phenotype

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