Yazarlar Lindsey Nash tekniğin geliştirilmesi ile ilgili yardım için teşekkür etmek istiyorum. Bu çalışma, Pittsburgh Üniversitesi, Pittsburgh OAIC Üniversitesi (AG024827) ve tohum fonları bir pilot proje hibe ALF, Ulusal Sağlık Enstitüsü, hibe AG028977 tarafından finanse edildi.

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Fosmids gelen transgenlerin nesil ana organizatörü elemanları, splice varyantları ve 3 &#39;UTR düzenleyici elemanlar tüm istinat yararına sunmaktadır. Bu yerli ifade desen, ya da diğer yaklaşımlar başarısız 5 işlevsel bir transgen inşaat daha yansıtıcı bir transgen inşaat yol açabilir . Ortaya çıkan transgenlerin GFP veya TAP etiketi epitopu etiketleri de dahil olmak üzere çeşitli taşıyabilir. Transgenlerin inşaatı tüm SW106 bakteriyel leke 15<...

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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

Transgenik hayvanların yaratılması, yaygın olarak C. kullanılmaktadır elegans araştırma, düzenleme ve faiz veya tandem afinite arıtma nesil (TAP) belirli genlerin sürümlerini kendi arıtma kolaylaştırmak için etiketli genlerin ifade desen çalışma GFP füzyon proteinleri kullanımı da dahil olmak üzere . Genelde transgenlerin GFP muhabiri gen ya da ilgi cDNA yukarı organizatörü koyarak üretilir ve bu genellikle temsil eden bir ifade kalıbı üretmektedir. Ancak, gen regülasyonu, 3 &#39;çevrilmemiş bölge ya da alternatif rehberleri kontrol elemanları gibi kritik unsurlar, bu yaklaşım tarafından kaçırılmış olabilir. Ayrıca sadece tek bir ek çeşidi genellikle bu yolla incelenebilir. Aksine, fosmid DNA klonları tarafından yapılan solucan genomik DNA muhtemelen içerir, en gerçek ifade desen ve zamanlama yakalamak için daha fazla yetenek izin in vivo gen regülasyonu ilgili tüm unsurlar değilse . Fosmid DNA kullanarak transgenlerin üretimi kolaylaştırmak için, E. tarif coli gen herhangi bir yere GFP, TAP-etiket veya ilgi diğer dizileri eklemek için recombineering prosedür. Bu prosedür, yüksek verimlilik ile istenilen değişiklik elde sonuçlar recombineering pozitif ve negatif seçim adımları hem galK gen seçim belirteci olarak kullanır . Ayrıca, yaygın olarak kullanılan GFP homoloji kolları ile çevrili ve füzyon genleri TAP galK genini içeren plazmid GFP veya TAP füzyon proteini oluştururken,% 50 oranında oligos maliyeti azaltmak mevcuttur . Bu plazmid geniş PCR ürün arıtma için ihtiyaç engellemektedir R6K çoğaltma kökenli kullanın. Nihayet, biz de unc-119 işaretleyici fosmid doğrudan enjekte veya transgenik hayvanlar üretmek için solucanlar içine bombardıman olanak sağlar fosmid omurgasına entegre bir teknik göstermektedir. Bu video bu yöntemi kullanarak recombineering üzeri transgen üreten yer alan usul göstermektedir.

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

Için transgenlerin üretme yeteneği<em&gt; Caenorhabditis elegans</em&gt; Tüm yerli düzenleyici unsurların korunur fosmids tarafından yapılan genomik DNA kullanarak özellikle çekici. Transgenlerin üretimi için basit ve sağlam bir prosedür ile recombineering yoluyla. Açıklanan<em&gt; GalK</em&gt; Seçilebilir işaretleyici.

Üretimi C. elegans Recombineering yoluyla transgenlerin GalK Seçilebilir Marker

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 Görüntüleme.  Beth Israel Deaconess Medical Center, Harvard Medical School. Için transgenlerin üretme yeteneği Caenorhabditis elegans Tüm yerli düzenleyici unsurların korunur fosmids tarafından yapılan genomik DNA kullanarak özellikle çekici. Transgenlerin üretimi için basit ve sağlam bir prosedür ile recombineering yoluyla. Açıklanan GalK Seçilebilir işaretleyici.