Gli autori desiderano ringraziare Lindsey Nash aiuto per sviluppare la tecnica. Questo lavoro è stato finanziato dal NIH concedere AG028977 di ALF, una borsa di progetto pilota presso l&#39;Università di Pittsburgh OAIC (AG024827), e fondi presso l&#39;Università di Pittsburgh.

<ol>
	<li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Zhang, Y., Nash, L., Fisher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified,+robust,+and+streamlined+procedure+for+the+production+of+C.+elegans+transgenes+via+recombineering.">A simplified, robust, and streamlined procedure for the production of C. elegans transgenes via recombineering.</a> BMC Dev Biol. 8, 119-119 (2008).</li><li>Antebi, A., Yeh, W. H., Tait, D., Hedgecock, E. M., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-12+encodes+a+nuclear+receptor+that+regulates+the+dauer+diapause+and+developmental+age+in+C.+elegans.">daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans.</a> Genes and Development. 14, 1512-1527 (2000).</li><li>Snow, M. I., Larsen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+expression+of+daf-12:+a+nuclear+hormone+receptor+with+three+isoforms+that+are+involved+in+development+and+aging+in+Caenorhabditis+elegans.">Structure and expression of daf-12: a nuclear hormone receptor with three isoforms that are involved in development and aging in Caenorhabditis elegans.</a> Biochim. Biophys. Acta. 1494, 104-116 (2000).</li><li>Fisher, A. L., Page, K. E., Lithgow, G. J., Nash, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+K10C2.4+Gene+Encodes+a+Member+of+the+Fumarylacetoacetate+Hydrolase+Family.+A+CAENORHABDITIS+ELEGANS+MODEL+OF+TYPE+I+TYROSINEMIA.">The Caenorhabditis elegans K10C2.4 Gene Encodes a Member of the Fumarylacetoacetate Hydrolase Family. A CAENORHABDITIS ELEGANS MODEL OF TYPE I TYROSINEMIA.</a> J Biol.Chem. 283, 9127-9135 (2008).</li><li>Wightman, B., Ha, I., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranscriptional+regulation+of+the+heterochronic+gene+lin-14+by+lin-4+mediates+temporal+pattern+formation+in+C.+elegans.">Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.</a> Cell. 75, 855-862 (1993).</li><li>Lehrbach, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIN-28+and+the+poly(U)+polymerase+PUP-2+regulate+let-7+microRNA+processing+in+Caenorhabditis+elegans.">LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans.</a> Nat Struct Mol Biol. 16, 1016-1020 (2009).</li><li>Tursun, B., Cochella, L., Carrera, I., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolkit+and+robust+pipeline+for+the+generation+of+fosmid-based+reporter+genes+in+C.+elegans.">A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans.</a> PLoS One. 4, e4625-e4625 (2009).</li><li>Bamps, S., Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+gene+expression+pattern+analysis,+in+situ,+in+Caenorhabditis+elegans.">Large-scale gene expression pattern analysis, in situ, in Caenorhabditis elegans.</a> Brief. Funct. Genomic. Proteomic. , (2008).</li><li>Dolphin, C. T., Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+reporter+fusion+genes+generated+by+seamless+modification+of+large+genomic+DNA+clones.">Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones.</a> Nucleic Acids Res. 34, e72-e72 (2006).</li><li>Sarov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombineering+pipeline+for+functional+genomics+applied+to+Caenorhabditis+elegans.">A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.</a> Nat. Methods. 3, 839-844 (2006).</li><li>Yang, X. W., Model, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+recombination+based+modification+in+Escherichia+coli+and+germline+transmission+in+transgenic+mice+of+a+bacterial+artificial+chromosome.">Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome.</a> Nat Biotechnol. 15, 859-865 (1997).</li><li>Court, D. L., Sawitzke, J. A., Thomason, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+using+homologous+recombination.">Genetic engineering using homologous recombination.</a> Annu.Rev.Genet. 36, 361-388 (2002).</li><li>Westenberg, M., Bamps, S., Soedling, H., Hope, I. A., Dolphin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+MW005:+lambda+Red-mediated+recombineering+and+copy-number+induction+of+oriV-equipped+constructs+in+a+single+host.">Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host.</a> BMC Biotechnol. 10, 27-27 (2010).</li><li>Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36-e36 (2005).</li><li>Penfold, R. J., Pemberton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+suicide+vector+for+construction+of+chromosomal+insertion+mutations+in+bacteria.">An improved suicide vector for construction of chromosomal insertion mutations in bacteria.</a> Gene. 118, 145-146 (1992).</li><li>Praitis, V., Casey, E., Collar, D., Austin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+low-copy+integrated+transgenic+lines+in+Caenorhabditis+elegans.">Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.</a> Genetics. 157, 1217-1226 (2001).</li><li>Puig, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 (2001).</li><li>Rigaut, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat.Biotechnol. 17, 1030-1032 (1999).</li><li>Achilleos, A., Wehman, A. M., Nance, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAR-3+mediates+the+initial+clustering+and+apical+localization+of+junction+and+polarity+proteins+during+C.+elegans+intestinal+epithelial+cell+polarization.">PAR-3 mediates the initial clustering and apical localization of junction and polarity proteins during C. elegans intestinal epithelial cell polarization.</a> Development. 137, 1833-1842 (2010).</li><li>Maduro, M., Pilgrim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+cloning+of+unc-119,+a+gene+expressed+in+the+Caenorhabditis+elegans+nervous+system.">Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system.</a> Genetics. 141, 977-988 (1995).</li></ol>

Nessun conflitto di interessi dichiarati.

La generazione di transgeni da fosmids offre il vantaggio di mantenere tutti gli elementi promotore nativo, varianti di splicing, e 3 &#39;UTR elementi di regolamentazione. Questo può portare alla costruzione di un transgene che è più riflettente del pattern di espressione nativa, o la costruzione di un transgene funzionale quando altri approcci non 5. I transgeni risultante può trasportare una varietà di etichette tra cui epitopo GFP o un tag TAP. La costruzione di transgeni...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FosmidMAX kit</td>
<td>&nbsp;</td>
<td>Epicentre</td>
<td>FMAX046</td>
<td>&nbsp;</td>
<tr>
<td>GoTaq</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>M7122</td>
<td>&nbsp;</td>
<tr>
<td>MOPS Media</td>
<td>&nbsp;</td>
<td>Teknova</td>
<td>M2120</td>
<td>&nbsp;</td>
<tr>
<td>0.132 M Potassium phosphate solution</td>
<td>&nbsp;</td>
<td>Teknova</td>
<td>M2102</td>
<td>&nbsp;</td>
<tr>
<td>D-galactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G0750</td>
<td>&nbsp;</td>
<tr>
<td>2-deoxygalactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4407</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4639</td>
<td>&nbsp;</td>
<tr>
<td>Leucine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L8000</td>
<td>&nbsp;</td>
<tr>
<td>NH4Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9434</td>
<td>&nbsp;</td>
<tr>
<td>Phusion DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>F-530S</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey agar base</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>281810</td>
<td>&nbsp;</td>
<tr>
<td>Arabinose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3131</td>
<td>&nbsp;</td>
<tr>
<td>Chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1919</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5136</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5655</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5886</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G2025</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>214010</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the production of c. elegans transgenes via recombineering with the galk selectable marker

La creazione di animali transgenici è largamente utilizzato in C. elegans ricerca compreso l&#39;uso di proteine ​​di fusione GFP per studiare la regolamentazione e pattern di espressione di geni di interesse o di generazione di purificazione in tandem affinità (TAP) tagged versioni di geni specifici per facilitare la loro purificazione. Tipicamente transgeni sono generati mettendo un promotore a monte di un gene reporter GFP o cDNA di interesse, e questo produce spesso un modello rappresentativo di espressione. Tuttavia, gli elementi critici della regolazione genica, come elementi di controllo nella regione non tradotta 3 &#39;o promotori alternativa, potrebbe non essere raggiunto da questo approccio. Inoltre solo una variante di splicing singolo può essere in genere studiati con questo mezzo. Al contrario, l&#39;uso del DNA genomico verme portato da cloni di DNA fosmid comprende probabilmente la maggior parte se non tutti gli elementi coinvolti nella regolazione genica in vivo che permette la maggiore capacità di catturare il modello autentico di espressione e la tempistica. Per facilitare la generazione di transgeni usando il DNA fosmid, ci descrivono un E. coli procedura basata recombineering per inserire GFP, un TAP-tag, o sequenze di interesse in qualsiasi posizione nel gene. La procedura utilizza il gene galK come marcatore di selezione per entrambe le fasi di selezione positiva e negativa in recombineering che si traduce ad ottenere la modifica desiderata con alta efficienza. Inoltre, plasmidi contenenti il gene galK affiancato da braccia omologia con GFP di uso comune e TAP geni di fusione sono disponibili che riducono il costo di oligo del 50% quando si genera un GFP o proteina di fusione TAP. Questi plasmidi utilizzare l&#39;origine replica R6K che esclude la necessità di ampia purificazione dei prodotti PCR. Infine, dimostrano anche una tecnica per integrare il unc-119 marcatore al backbone fosmid che permette fosmid di essere iniettato direttamente o bombardati in vermi per generare animali transgenici. Questo video mostra le procedure coinvolte nella generazione di un transgene attraverso recombineering utilizzando questo metodo.

Watch this Scientific Journal Video about The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker at JoVE.com

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

La capacità di produrre transgeni per<em&gt; Caenorhabditis elegans</em&gt; Usando il DNA genomico portato da fosmids è particolarmente interessante come tutti gli elementi nativi normativo vengono conservati. Descritto è un procedimento semplice e robusto per la produzione di transgeni attraverso recombineering con il<em&gt; GalK</em&gt; Marcatore.

La produzione di C. elegans Transgeni attraverso recombineering con il GalK Marcatori selezionabili

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

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The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

14.5K Views.  Beth Israel Deaconess Medical Center, Harvard Medical School. La capacità di produrre transgeni per Caenorhabditis elegans Usando il DNA genomico portato da fosmids è particolarmente interessante come tutti gli elementi nativi normativo vengono conservati. Descritto è un procedimento semplice e robusto per la produzione di transgeni attraverso recombineering con il GalK Marcatore.