The mammalian gene function resource: the International Knockout Mouse Consortium.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research

The mammalian gene function resource: The International Knockout Mouse Consortium

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed highthroughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research

Ribonuclease activity and RNA binding of recombinant human Dicer

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated ∼21–23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer·dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes

Overexpression of the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>^<i>+</i> gene suppressed exon skipping in <i>ods4</i>-<i>1</i>.

<p>(A) The overexpression of the <i>cwf16</i>⁺ or <i>tif213</i>⁺ gene rescued the <i>cs</i> phenotype of <i>ods4-1</i>. Transformants with pBG1-URA4β and the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>^<i>+</i> plasmid were streaked on MMU plates and incubated at 22, 26, or 30°C to test their complementarity for the <i>cs</i> phenotype of <i>ods4-1</i>. Transformants with the <i>cwf16</i>^<i>+</i> or <i>tif213</i>^<i>+</i> plasmid grew well at 22°C, whereas <i>ods4-1</i> itself showed slow growth at the same temperature (<i>ods4</i>+URA4β+pSP1). The overexpression of Srp2p resulted in slow growth at all temperatures. (B) Total RNAs were isolated from wild type (WT), <i>ods4-1</i>, and <i>ods4-1</i> with the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>⁺ plasmid, and subjected to RT-PCR analyses. All transformants contained pBG1-URA4β in addition to the rescued genes or pSP1 vector as indicated. Amplified cDNA products were electrophoresed on a 5% acrylamide gel, stained with ethidium bromide (upper panel), and then subjected to a Southern blot analysis using an oligonucleotide probe that specifically hybridizes to the exon-skipped product (middle panel). RT-PCR of <i>act1</i>^<i>+</i> mRNA was performed as a control (lower panel). The structures of the RT-PCR products confirmed by the sequence analysis are shown on the left. Arrows indicate the positions of the tub-3 and tub-4 primers used for RT-PCR analyses.</p

Structure of reporter plasmids for <i>ods</i> screening.

<p>(A) Structures of pURA4β (pSP1-URA4β) and pBG1-URA4β reporter plasmids. The pSP1 and pBG1 vectors have the <i>LEU2</i> and <i>his3</i> markers, respectively. The intron 1-exon 2-intron 2 region of the <i>S</i>. <i>pombe</i> β-tubulin gene (<i>nda3</i>^<i>+</i>) was amplified by PCR and inserted into the <i>Stu</i> I site in the <i>ura4</i>^<i>+</i> gene. (B) Splicing patterns of the transcripts from the chimeric <i>ura4</i> gene in the reporter plasmids. Transcripts in which the internal exon is included produce non-functional Ura4p, whereas exon-skipped transcripts produce functional Ura4p.</p

List of oligonucleotides.

<p>List of oligonucleotides.</p