Yazarlar mikroenjeksiyon yanı sıra anonim değerlendirenler anlayışlı görüşleri ile ilgili yardım için RJ Sommer ve X Wang müteşekkiriz. Bu çalışma NIH hibe SC2GM089602 tarafından desteklenmektedir.

1. Transgenesis: DNA hazırlığı <ol><li> Hakim ko-enjeksiyon belirleyicisi: pRL3 [Ppa-PRL-1 (tu92)] PRL3 plazmid görsel olarak başarılı bir dönüşüm olayları tanımlamak için, baskın bir ko-enjeksiyon belirteç . Bu plazmid yakından C. SQT-1 kolajen geni ile ilgili Ppa-PRL-1 geni baskın bir mutant allel (tu92) için kodlar elegans ve wild tip sinüzoidal lokomosyon solucan vücut ekseni 1,5 boyunca saat yönünde büküm hareketleri haline dönüştürür. Dönüştürülmüş hayvan C. kullanılan popüler bir baskın seçim işaretleyici rol-6 (su1006) çok benzer. Geçtiğimiz 20 yıl boyunca elegans. </li><li> Hedef muhabiri transgen: Ppa-egl-4p: GFP Ilgi bir gen dizisi 9 kodlama GFP transkripsiyonel veya translationally bağlantılı olabilir. Bu çalışmada, Ppa-egl-4promoter: GFP (egl-4, cGMP bağımlı protein kinaz) kullanımıçeviri başlangıç ​​upstream 2 kb parçası S. GFP ifade kazandırmaz eğer d belirlemek için pacificus PS312. Bu GFP vektör modifiye ile GFP kodlama bölgeyi içeren intron ve çok amaçlı Ppa-rpl-23 nazik Xiaoyue Wang ve Ralf J. Sommer (Max-Planck Enstitüsü, Tübingen, Almanya, AB) tarafından sağlanan 3 &#39;UTR terminatör 5. </li><li> Genomik DNA: PS312 Ayrıca yabani-tip P. pacificus PS312 genomik DNA, özellikle transgenik F 1, F 2 döl 5 hayvanlar, istikrarlı transgen miras almak için gereklidir . Kararlı F 2 transgenik hatları görülenlere benzer: genomik DNA iki transgenlerin (GFP muhabiri: ko-enjeksiyon işaretleyici PRL-3 ve Ppa-egl-4p) ile karmaşık ekstra kromozom dizileri oluşturur henüz belli değil C. elegans 3. Ancak, aynı cohesi gereksinimi paylaşmak için genomik DNA ve transgenlerin varve çıkıntılar, güçlü konak hücrelerine doğru DNA şanzıman (gDNA Sigma G1N10 kiti kullanılarak hazırlanmıştır) tanıyabilir karmaşık ekstra-kromozomal dizileri oluşumunu göstermektedir. </li><li> DNA sindirimi : GFP vektör DNA ve ko-enjeksiyon işaretleyici pRL3 ve PS312 gDNA uyumlu yapışkan çıkıntılar üretmek için: Kısıtlama enzimler Ppa-egl-4p linearize için kullanılır. Vorteks değil, dokunarak karıştırın. 37 az bir saat boyunca inkübe ° C </li></ol><table border="1"><tbody><tr><td> pRL3 </td><td> 4 mikrogram </td></tr><tr><td> 10xbuffer </td><td> 10 ul </td></tr><tr><td> PstI (10 U / ml) </td><td> 4 ul </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Toplam: </td><td> 100 mcL </td></tr><tr><td> Ppa-egl-4p: GFP </td><td> 4 mikrogram </td></tr><tr><td> 10x tampon </td><td> 10 ul </td></tr><tr><td> Sali (10 U / ml) </td><td> 4 ul </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Toplam: </td><td> 100 ul </td></tr><tr><td> gDNA PS312 </td><td> 10 mikrogram </td></tr><tr><td> 10x tampon </td><td> 10 ul </td></tr><tr><td> PstI&#39;in ve Sal I (10 U / ml) </td><td> Ul 8 </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Toplam: </td><td> 100 ul </td></tr></tbody></table> Tablo 1 Kısıtlama Enzim Digest için karışım. <ol start="5"><li> DNA yağış ve hazırlık <ol><li> 1:10 sodyum asetat (3 M NaCOOCH-3 pH 5.2) ve sindirilmiş ürün% 100 etanol hacmi 2.5X ekle, pelet daha kolay görmek için 20 ng / glikojen ul ekleyin . </li><li> 15 dakika 14.000 rpm&#39;de Santrifüj 4 ° C </li><li> Tippi tarafından süpernatant Durusuyavaş yavaş ng santrifüj tüpü baş aşağı ve daha sonra bir kağıt mendil, dokunarak, pelet, tüpün alt köşesindeki güvenli olduğundan emin ve etanol ücretsiz. </li><li> 1 ml% 75 etanol ekleyin. </li><li> Hemen 4 5 dakika 14.000 rpm az dönmeye ° C </li><li> Yavaş ters santrifüj tüpüne devrilme ve sonra bir kağıt mendil pelet bu tüpün alt köşesindeki güvenli ve emin etanol dokunarak süpernatant Durusu. </li><li> Kuru pelet 5 dakika 14.000 rpm&#39;de santrifüj vakum 25-30 ° C, kuru ve tamamen ücretsiz etanol kadar. </li><li> TE veya steril dH 2 O 30 ul ekleyin ve bir girdap ile 4 ° C karıştırma kısa bir süre önce bir gece bekletin. </li><li> Tablo 2, enjeksiyon karışımı enjeksiyon için her saflaştırılmış DNA nasıl oluşturulacağı anlatılmaktadır. </li><li> -20 ° C&#39;de muhafaza Enjeksiyon karışımı çözündükten sonra olabilir enkaz parçacıklar kurtulmak için tüp 5 dakika 14.000 rpm&#39;de aşağı doğru döndürünenjeksiyon iğnesi tıkayabilir. </li></ol></li></ol><table border="1"><tbody><tr><td> Genetik materyal </td><td> Konsantrasyon </td></tr><tr><td> Ppa-egl-4p: GFP (Sali) </td><td> &gt; 5 ng / ul (0.1-10ng/μl) </td></tr><tr><td> pRL3 (PstI) </td><td> &gt; 1 ng / ml </td></tr><tr><td> gDNA PS312 (PstI + Sali) </td><td> &gt; 60 ng / ml </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Toplam: </td><td> 30 ul </td></tr></tbody></table> (Tablo 2). Ppa-PRL-1 enjeksiyon karışımı 2. RNA Girişim: in vitro çift zincirli RNA sentezi <ol><li> PCR ilgi gen yükseltmek için kullanın. RHL091 5&#39;-agtggatccGAAGGTCCATACGGGAGC 3 &#39;(BamHI site) ve RHL092 5&#39;-tatctgcagGTGAGGAGTACCAGGAGAG-3&#39; (PstI site) 464 bp genomik frag yükseltmek için kullanılır.Ppa-PRL-1 (tu92) aleli içeren pRL3 vektör Ment (konum 3-466). </li><li> BamHI ve PstI ile PCR ürünü Digest. </li><li> BamHI / PstI pL4440 RNAi besleme vektör 2 sindirilir parçası Arter. </li><li> Yetkili hücrelerin içine eklenen vektör Dönüşümü ve Ampisilin LB medya levhalar (50 mg / ml) onları büyümek. </li><li> T7 primerler kullanılarak PCR ile başarıyla takılı vektör seçin. </li><li> BLOK-IT RNAi sentezi kiti (Invitrogen) kullanarak) çift iplikli RNA dsRNA yapmak için PRL-1 geni içeren PCR ürünü çevrili T7 kullanın. </li><li> Kadar 1 enjeksiyon için mikrogram / ul&gt; 200 ng / ml, dsRNA konsantrasyonu en iyi. </li><li> -20 ° C&#39;de muhafaza Enjeksiyon karışımı çözündükten sonra, bir iğne tıkayabilir enkaz parçacıkların kurtulmak için tüp 5 dakika 14.000 rpm&#39;de dönerler. </li></ol> 3. Mikroenjeksiyon: transgenlerin ve / veya dsRNA enjeksiyon Protokolü <ol><li> Enjeksiyon İğnesi <ol><li> Sıcaklığı: Narishige iğne çektirmesi (PC-10 model #) ayarlayın <ol><li> Alt düğmeyi çevirin &quot;No.2 Isıtıcı.&quot; </li><li> &quot;No.2 Isıtıcı Ayarı&quot; Turn. paneli kadar okur topuzu 55,0-60,0 ° C değişen uzunluklarda konik iğne ucu. </li></ol></li><li> (Bir adım iğne çeker) &quot;Adım 1&quot; geri altındaki düğmeyi çevirin. </li><li> Iğne odasına yükleyin ve güvenli olduğundan emin olun. </li><li> &quot;Başlat&quot; düğmesini (kırmızı düğme) basın ve iğne (bu iki zıt yönlerde aynı iğne yapmak gerekir) tüm ağırlıkları ile çekilmesine izin verir. </li><li> Iğne, iğneler üzerinde biriken tozu önlemek için Play-Doh ile sabitleniyor plastik bir kültür plaka oda ve mağaza çıkarın. </li></ol></li><li> Montaj Pedleri <ol><li> 1,5 ml santrifüj tüpüne, 1 ml H 2 O Noble Agar 0,02 g ekleyerek% 2 Noble Agar çözüm Agar, 4 ° C ile bir yıl kadar saklanabilir. </li><li> İyice karıştırın ve&gt; 88 ° C&#39;ye ayarlanmış bir ısı bloğu agar eritebilir </li><li> 1 ml mikropipet kullanarak, (Fisher, 12-544-F) sıvı% 2 Noble Agar bir damla cam slayt ortasına yerleştirin. </li><li> Damla üstünde başka bir cam slayt koyarak düzleştirin. </li><li> &quot;Bırak Adım&quot; Beş cam slayt bir katman kalmayıncaya kadar tekrarlayın. </li><li> Her cam sürgülü cam slayt çıkarın birbirlerine slaytlar. </li><li> Kuru bir elektrikli fırında 70 cam slaytlar agar ped düzleştirmek ° C gecede 4 saat (ya da oda sıcaklığında gece). </li><li> Ileride kullanmak için birden fazla Enjeksiyon pedleri ve mağaza olun. </li></ol></li><li> Hermaphroditic gonadlar içine mikroenjeksiyon </li></ol><img alt="Şekil 1" src="/files/ftp_upload/3270/3270fig1.jpg" /> Şekil 1. P. şematik çizim pacificus anatomi net çekirdekli hücreler dişi germ hücreleri (oosit) ve gri gölgeli kısmı bağırsak. Sadece bir distal anterior gonad gösterilirama iki distal gonadlar, gövde ortasında yakın dorsal birbirlerine çapraz dikkat edin. <img alt="Şekil 2" src="/files/ftp_upload/3270/3270fig2.jpg" /> Şekil 2. Görüntüleri mikroenjeksiyon, (A) enjeksiyon iğne ucu çekti kılcal parçasının aşırı sağ bir çizgi ile odak . Keskin bir nokta hala korurken, bu kenarı iğne ucu hafifçe dokunarak, iğne ucu ayrılmalıyız. (B) üst içine iğne ekler sadece ilk enjeksiyon için bağırsak eğriliği yukarıdaki gonaden. (C) altına iğne ekler ikinci enjeksiyon için sadece gut eğrilik altında gonaden. <ol start="3"><li><ol><li> Kontrast ve DIC kesme ayarı hermafrodit gonadlar görüntülemek için ideal olduğundan emin olun. </li><li> Çekti kılcal iğne içine enjeksiyon karışımı 1.0-2.0 ul yükleyin. </li><li> Mikroenjeksiyon manipülatör iğne yerleştirin; nitrojen tankı basınç göstergeleri ideal koşulları belirlenmiş olmalıdır (10-20 psi0.5-1 saniye süreyle). </li><li> Iğne ucu bir slayt (Şekil 2A) yerleştirilen, ince çekti kılcal tüp kenarına hafifçe dokunarak kırın. <ol><li> İğne kesici slayt çekti petrol bir damla cam bir slayt üzerine yerleştirilen kapiller ince parçası </li></ol></li><li> İğne sonra (P. pacificus onlar kuru önce enjeksiyon için 5 dakikalık bir pencere var ve ölmek) pad solucan almak ise atıl bir konumda yer olabilir. </li><li> J4-genç erişkin solucanlar dolu bir tabak kullanarak, tüm solucanlar OP50 bakteriyel çim kapağı sadece yeterli parafin yağı (ağır) dökün. </li><li> Pick bir solucan ve enjeksiyon pad üzerine yerleştirin. </li><li> Iğne ve iğne (Şekil 2B, 2C) uzak vulva yönde pozisyon solucan gonad mikroskop solucan ile cam slayt yerleştirin. </li><li> Mikroskop görünümü konumu iğne indirin. </li><li> Aynı odak düzlemi (Şekil 2B) üst gonad ve iğne yerleştirin.</li><li> Yavaşça solucan Poke ve pompa enjeksiyon karışımı; distal uç doğru ayıran ooctyes (transgen veya dsRNA teslim optimize etmek için başarılı bir enjeksiyon gösterir, enjeksiyon üzerine distal yakın olana kadar devam gonad genişleme dalgası görmelisiniz ucu). </li><li> Yeniden konumlandırmak alt gonad ve tekrar enjeksiyon enjekte etmek için iğne, bu gonad oositlerin yayılması olmayacaktır bağırsak altında ve (Şekil 2C) ulaşmak için böylece daha zor ancak. </li><li> Normal görünümlü solucan enjeksiyondan sonra başarılı bir enjeksiyon gösterir; (vulva dışarı çıkıntılı ya da vücut boşluğunun içine attı) herhangi bir hasarlı gonadlar olmamalıdır. </li><li> Rölanti iğne geri yerleştirin. </li><li> Solucan kurtarmak için hazırlayın. </li><li> M9 tampon enjekte solucan yerleştirilen ped üzerine bir damla (0.5 ul) ekleyin. </li><li> OP50 bakteri tercihinizi kullanarak, solucan OP50 ile dokunma, ve sopa olmalıdır. We, ağız pipeti kullanmayın. </li><li> Solucan tabağa OP50 50 ul noktalar ile seeded yerleştirin, 2-4 solucanlar gibi plakalar kurtarıldı olabilir. </li></ol></li><li> Transgenesis için enjeksiyon sonrası Depolama ve Tarama <ol><li> Mağaza 20 (P 0) solucanlar enjekte ° C ~ 4 gün yumurtalarını ve F 1 büyüyecek. </li><li> Seçili fenotip (transgenesis Ppa-PRL-1 hakim işaretleyici ifade yuvarlanan hayvanlar için, arama ekran) için dördüncü gününde Ekran solucanlar. </li><li> Tek bağımsız F 1 Pick 25 yeni numaralı seribaşı plaka ve saklamak için gözlenen bir fenotip ile hayvanlar ° C; bu sıcaklık F 1 istikrarlı çizgilerin oluşumunu teşvik eder &#39; s. </li><li> Tek F 2 F 1 hatlarından bağımsız. Bireysel F 2 satır benzer transgen aktarım hızlarına sahip. Biz genellikle&gt; 15 elde F dönüştürülmüş F 1 hayvan başına 2 satır. </li><li> 25 ° C&#39;de sonraki nesillere Mağaza </li><html><li> Her bir hat için: silindirleri iletim hızı genellikle bu hedef transgen ekspresyon seviyesi (GFP: Ppa-egl-4p) belirlemek için şu anda gerekli olan F 4 nesil sonra sabit kalır. GFP ifade baskın rulo fenotip penetrans derecesi ile ilişkili olmayabilir. </li></ol></li><li> RNAi fenotipleri için enjeksiyon sonrası Depolama ve Tarama <ol><li> Mağaza 20 (P 0) solucanlar enjekte ° C ~ 4 gün yattı ve F 1 büyümek için. </li><li> Ekran F seçilen fenotip (RNAi demonte fenotipi Ppa-PRL-1 fenotip kaybı bizim durumumuzda, hedef gen aktivitesinin kaybı ile ilişkili olabilir penetrant fenotipleri için arama için ekran) dördüncü gününde 1 solucanlar. </li><li> Deneyimlerimize göre, non-silindiri demonte fenotip F 2&gt;% 97 kaybolur. </li></ol></li></ol> 4. Temsilcisi sonuçları: <html> Transgenesis p &quot;&gt; 1 Sonuç <table border="1"><tbody><tr><td> Oturum </td><td> P 0 Injected </td><td> F 1 Silindirler </td><td> % Transgenik F 2 </td><td> Ortalama% iletim F 2 sonra </td></tr><tr><td> 1 </td><td> 60 </td><td> 2 </td><td> (Hat 1) 0% (2 hat)% 13 </td><td> NA % 13 ± 10 </td></tr><tr><td> 2 </td><td> 40 </td><td> 1 * </td><td> (3 hat)% 0 </td><td> NA </td></tr><tr><td> 3 </td><td> 40 </td><td> 0 </td><td> % 0 </td><td> NA </td></tr><tr><td> 4 </td><td> 40 </td><td> 1 </td><td> (4 hat)% 0 </td><td> NA </td></tr><tr><td> 5 </td><td> 20 </td><td> 0 </td><td> % 0 </td><td> NA </td></tr><tr><td> 6 </td><td> 40 </td><td> 1 </td><td> % 26 (5 hat) </td><td> % 22 ± 10 </td></tr></tbody></table>içeriği &quot;&gt; çapraz vermedi * makaralı bir erkek; NA: Uygulanabilir değil : GFP (Sal I sindirilir), pRL3 (rulo) (PstI&#39;in sindirilir) ve gDNA PS312 (PstI ve Sali sindirilir): Tablo 3 Sonuçları Ppa-egl-4p enjekte PS312. <img alt="Şekil 3" src="/files/ftp_upload/3270/3270fig3.jpg" /> Şekil 3. (A) wild tip (B, C) ​​istikrarlı bir F 4 pRL3; Ppa-egl-4p:: nöron GFP ifade baş gösteren GFP transgenik hattı. 2. RNA girişim sonucu <img alt="Şekil 4" src="/files/ftp_upload/3270/3270fig4.jpg" /> Şekil 4. RNA PRL-1 mutantlar &quot;yuvarlanan&quot; hareketin komple demonte (A) PRL-1 rulo kazanç fonksiyon-mutant tu92 enjekte etti. (B) PRL-1 mutant sergi, normal wild tip duruş ve hareket &quot;nakavt&quot;. (C) PRL-1 (alt) da uzun b Injecteddaha ody PRL-1 mutant (üstte). (D) uzun vücut enjekte PRL-1 (alt) (üst) wild tip PS312 daha uzun da. Uzun vücut fenotip aynı zamanda rol-5 C. (SQT-1) RNAi demonte gözlendi elegans N2 (veriler gösterilmemiştir). <table border="1"><tbody><tr><td></td><td> rulo </td><td> non-roll </td></tr><tr><td> Bir PRL-1 dsRNA (200 ng / ul) </td><td> 13 </td><td> 21 </td></tr><tr><td> Yatak PRL-1 dsRNA (ng / ul 1000) </td><td> 9 </td><td> 16 </td></tr><tr><td> kontrol </td><td> 20 </td><td> 2 </td></tr></tbody></table> Tablo 4 Ppa-PRL-1 dsRNA enjeksiyonları özeti. (A) [dsRNA] = 200 ng / ul; (B) [dsRNA] = 1000 ng / ul. P = 0.0019 ve 0.041, sırasıyla 200 ve 1000 ng / ul enjeksiyonları için iki kuyruklu Fisher Exact Testi,,.

<ol>
	<li>Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> Wormbook. , (2006).</li><li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.+elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10, 3959-3970 (1991).</li><li>Junio, A. J., Li, X., Massery, H. C., Nolan, T. J., Lamitina, S. T., Sundaram, M. V., Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongyloides+stercoralis:+cell-+and+tissue-specific+transgene+expression+and+co-transformation+with+vector+constructs+incorporating+a+common+multifunctional+3'+UTR.">Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR.</a> Exp. Parasitology. 118, 253-265 (2008).</li><li>Schlager, B., Wang, X., Braach, G., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+dominant+roller+mutant+and+establishment+of+DNA-mediated+transformation+in+the+nematode+Pristionchus+pacificus.">Molecular cloning of a dominant roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus.</a> Genesis. 47, 300-304 (2009).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pristionchus+pacificus:+a+well-rounded+nematode.">Pristionchus pacificus: a well-rounded nematode.</a> Bioessays. 28, 651-659 (2006).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattraction+in+Pristionchus+nematodes+and+implications+for+insect+recognition.">Chemoattraction in Pristionchus nematodes and implications for insect recognition.</a> Curr. Biol. 16, 2359-2365 (2006).</li><li>Rudel, D., Riebesell, M., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadogenesis+in+Pristionchus+pacificus+and+organ+evolution:+development,+adult+morphology+and+cell-cell+interactions+in+the+hermaphrodite+gonad.">Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad.</a> Dev. Biol. 277, 200-221 (2005).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 11, 802-805 (1994).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Worm+Lab:+from+Culturing+Worms+to+Mutagenesis.">An Introduction to Worm Lab: from Culturing Worms to Mutagenesis.</a> J. Vis. Exp. (47), e2293-e2293 (2011).</li><li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+and+Microinjection.">Transformation and Microinjection.</a> Wormbook. , (2006).</li></ol>

Çıkar çatışması ilan etti.

P. pacificus popülasyonları dünya çapında çeşitli bokböceği böcek türleri ile yakın ilişkisi bulunan ve serbest yaşayan ve paraziter nematod arasındaki ara bir model nematod . P. gücü gelişmekte olan bir model organizma olarak pacificus tarafsız ileri genetik ekranlarını izole mutantlar teşvik pozisyonel haritalama, genetik ve fiziksel haritalar entegrasyonu yatıyordu (yani sadece daha önce C. elegans karakterize aday genler için) 6,10. Anc...

<table border="1"><tbody><tr><th> Ürün: </th><th> Katalog #: </th><th> Faaliyet alanı: </th></tr><tr><td> DMI3000 Enjeksiyon mikroskop </td><td> DMI3000 </td><td> Leica </td></tr><tr><td> Mikroenjektör manipülatör (Direct drive) </td><td> Narashige BC-3 Rotil </td><td> TriTech Araştırma </td></tr><tr><td> İğne Çektirme </td><td> Narashige PC-10 </td><td> TriTech Araştırma </td></tr><tr><td> Mikroenjektör ™ All-Dijital Multi-basınç sistemi </td><td> Narashige MINJ-D </td><td> TriTech Araştırma </td></tr><tr><td> GeneElute ™ Memeli Genomik DNA Miniprep Seti </td><td> G1N70 </td><td> Sigma </td></tr><tr><td> pJet Klonlama Jet kiti </td><td> K1231 </td><td> Fermentas </td></tr><tr><td> GeneJET plazmid Miniprep kiti </td><td> K0502 </td><td> Fermentas </td></tr><tr><td> DNA Clean &amp; Konsantratörü ™ </td><td> D4005 </td><td> ZYMO Araştırma </td></tr><tr><td> BLOK-IT ™ RNAi TOPO ® transkripsiyon kiti </td><td> K3500-01 ve K3650-01 </td><td> Invitrogen </td></tr><tr><td> Difco ™ Agar Noble </td><td> DF0142-15-2 </td><td> Fisher bilimsel </td></tr><tr><td> Mikroskop cam kapak (1.5 - 0,16 - 0,19 mm kalınlığında; Boyut: 50 x 45mm) </td><td> 12-554-F </td><td> Fisher Scientific </td></tr><tr><td> Cam kılcal damarlar (filament) </td><td> 615000 </td><td> AM sistemleri </td></tr><tr><td> Parafin Yağı (Ağır) </td><td> O122-1 </td><td> Fisher Scientific </td></tr><tr><td> KH 2 PO 4 </td><td> P386-500 </td><td> Fisher Scientific </td></tr><tr><td> Na 2 HPO 4 </td><td> AC20651-5000 </td><td> Fisher Scientific </td></tr><tr><td> NaCl </td><td> BP3581 </td><td> Fisher Scientific </td></tr><tr><td> MgSO 4 </td><td> M80-500 </td><td> Fisher Scientific </td></tr></tbody></table>

rnai mediated gene knockdown and transgenesis by microinjection in the necromenic nematode pristionchus pacificus

RNA girişim ve istikrarlı transgenesis gen fonksiyonu ve ifade analizleri derinlemesine daha fazla, tamamen sıralı genomların elde birçok türün özellikle anatomi ve organizmanın moleküler hücre biyolojisi nedeniyle sınırlı kalmaktadır. Gelişmekte olan model organizmalar için gittikçe daha uygun fiyatlı olmasına rağmen Örneğin, stably Caenorhabditis olmayan türlerin transgenik hatları bulaşan Caenorhabditis elegans 3, taç grup Caenorhabditis dışında Strongyloides stercoralis 4 ve Pristionchus pacificus 5 istisna ile, bu aldatıcı filumun (Nematoda) bildirilmiştir değil. P. genişleyen rolünü kolaylaştırmak için çalışmada, gelişme, evrim, ve davranış 6-7 pacificus, biz burada, transgenik hayvanlar ve gen RNAi tarafından yıkmak yapmak için mikroenjeksiyon kullanmak için geçerli yöntemleri açıklanmaktadır. Gonadlar gibi <em&gt; C. elegans ve diğer birçok nematod, P. gonadlar pacificus syncitial ve doğrudan mikroenjeksiyon tarafından teslim oosit içine DNA ve RNA içeren yeteneğine sahip. C. aksine P. de elegans Ancak, istikrarlı transgen miras ve somatik ifade pacificus hedef transgenlerin ve coinjection belirteçler 5 biter tamamlayıcı endonükleaz ile sindirilir kendini genomik DNA eklenmesini gerektirir. Ayrıca taşıyıcı genomik DNA Strongyloides stercoralis 4 ve C germ hücreleri transgen ifade gereksinimi için benzer elegans. Ancak, bu hayvanların kendi genomik DNA için spesifik bir gereklilik ise açık değildir, çünkü P. pacificus soma karmaşık olmayan çok kopyalı genlerin susturulması ya da çok verimli P. içinde ekstrakromozomal diziler pacificus mitoz sırasında uygun kinetochore montaj için genomik dizileri gerektirir. Iki kollu (ventral göççift ​​cinsiyetli olarak didelphic) gonadlar, bireysel solucanlar 8 hem de gonadlar enjekte yeteneği daha da zorlaştırmaktadır. Ayrıca demonte haddeleme olmayan F 1 döl elde etmek için çift iplikli RNA (dsRNA) gonadlar içine enjekte edilerek baskın bir mutant (rulo, tu92) mikroenjeksiyon kullanımı göstermektedir. C. aksine elegans, ancak diğer pek çok nematod gibi, P. pacificus PS312 besleme ve iliklerine ve bu nedenle dsRNA syncitial gonadlar içine mikroenjeksiyon tarafından uygulanmalıdır yoluyla sistemik RNAi açık değildir. Bu çalışmada, biz Ppa-egl-4 organizatörü dönüştürmek için gerekli olan mikroenjeksiyon işlemi tanımlamak için umut: GFP füzyon muhabir ve demonte baskın bir rulo PRL-1 (tu92) mutant görsel bilgilendirici bir protokoldür.

Watch this Scientific Journal Video about RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus at JoVE.com

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

RNAi spesifik mRNA transkript 1-2 demonte model organizmalar, transgenesis gen işlevleri işleyebilirsiniz. Bu protokol, necromenic nematod içine stably iletilen DNA ve geçici çift iplikli RNA tanıtmak için gerekli teknikleri göstermek için hedefliyor<em&gt; Pristionchus pacificus</em&gt;, Evrimsel gelişim ve davranış biyolojisi çalışmaları için.

RNAi Necromenic nematodu Mikroenjeksiyon Gen demonte ve Transgenesis aracılığı ile Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

rnai-mediated-gene-knockdown-transgenesis-microinjection-necromenic

Research

JoVE Journal

Biology

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

16.4K Görüntüleme.  California State University. RNAi spesifik mRNA transkript 1-2 demonte model organizmalar, transgenesis gen işlevleri işleyebilirsiniz. Bu protokol, necromenic nematod içine stably iletilen DNA ve geçici çift iplikli RNA tanıtmak için gerekli teknikleri göstermek için hedefliyor Pristionchus pacificus, Evrimsel gelişim ve davranış biyolojisi çalışmaları için.

Video: RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Lokalize RNAi ve Tıbbi Sülük Ektopik Gen İfadesi

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Biyoloji

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Gen Fonksiyon Analiz Zebra balığı Embriyolar Mikroenjeksiyon

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNAi Efektör Bakteriyel Teslim Tarihi: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Sitokrom Sitoplazmik Mikroenjeksiyon Apoptoz aktivasyonu C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral aracılı demonte sırasında Ex vivo İnsan Hematopoetik Kök Hücre Eritropoiesis

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Pamuk olarak Agrobacterium Virüs Bağlı Gen susturma Testi

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-aracılı Gene Nakavt ve<em&gt; In Vivo</emYetişkin Kadınlar&gt; diürez Testi<em&gt; Aedes aegypti</em&gt; Sivrisinekler

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genom çapında Gen Delesyonlar içinde<em&gt; Streptococcus sanguinis</em&gt; Yüksek Verimli PCR ile

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Nematod Vücut büyüklüğü Yönetmelik ilgilendim Genler için Ekran RNA-aracılı Girişim Besleme Strateji Kullanımı<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...