Yazarlar cömertçe H1 MKH, ve Dr. Tim Hacker, Dr. Gouqing Song ve Wisconsin Kardiyovasküler Fizyoloji Üniversitesi Bayan Jill Koch sağlamak için Dr. Peiman Hemmati (Tıp Fakültesi, University of Wisconsin-Madison) teşekkür etmek istiyorum Fare cerrahisine Core Tesisi. Bu çalışma, Ulusal Bilim Vakfı tarafından Brian Freeman ve NIH R21 HL089679 Yüksek Lisans Araştırma Bursu ile desteklenmiştir.

1. Donör Hücre Transfeksiyon <ol><li> Hasat mezenkimal kök hücreler (MKH, lütfen Dr. Peiman Hematti tarafından bağışlanan H1, embriyonik kök hücrelerinden elde edilen alternatif olarak, in vivo olarak sigorta varsayımla herhangi bir türün herhangi bir hücre tipi istihdam edilebilir) 70 -% 80 1X tripsin ile konfluent (Mediatech 5 dakika için Manassas VA) α-MEM tam orta (ücretsiz, antibiyotik Invitrogen, Carlsbad CA) 18 tripsin İnaktivasyon. 5 dakika boyunca 300 xg&#39;de santrifüjleyin. </li><li> Dikkatle supernatant aspirat ve yeniden askıya pelet 1X PBS 1 mL ve bir hemasitometre kullanarak hücre sayımı. </li><li> 1.5 mL santrifüj tüpüne transfer 1,5 x 10 6 hücre. 5 dakika boyunca 300 xg&#39;de santrifüjleyin. </li><li> Dikkatle supernatant aspire. R Buffer (Neon Transfeksiyon Sistemi, Invitrogen) ve 6 mg (2 mcg / 5,0 x 10 5 hücre) 300 mcL yeniden süspanse pelet P231 pCMVe betaAc-STOP-luc (Addgene, Cambridge, MA). E Tampon Yeri 3 mL (Invitrüreticinin protokol başına elektroporasyon yerleştirme port (Neon Transfeksiyon Sistemi, Invitrogen) içine ogen). </li><li> 100 mcL Neon pipetlemeyin ucu hücre plazmid çözüm aktarın ve bir darbe süresi 20 ms ve 1500 volt bir büyüklük ile electroporate. 9.7 mL α-MEM tam orta içeren 15 ml konik bir tüp içine yerleştirin hücreleri electroporated. </li><li> 10 ml toplam hacmi verim 1.5 iki kez ve havuz transfekte hücreler adımı yineleyin. 10 ml tam orta α-MEM içeren bir T175 balona 10 ml hücre süspansiyonu (1.5 x 10 6 hücre) ekleyin. Elektroporasyon sonra hücre canlılığı, T175 başına yaklaşık 4,5 x 10 5 canlı hücreleri verim, yaklaşık% 30&#39;dur . </li><li> Değişim transfeksiyon şu ortamda 24 saat α-MEM tam. </li><li> % 80 konfluent (~ 2 - - 3 gün sonra elektroporasyon) Hasat 70 hücreleri transfekte. Bir hemasitometre kullanarak hücre sayımı gerçekleştir ve 1.0 x 10 6 bir konsantrasyon hücreleri tekrar süspansiyonα-MEM tam orta cells/50 mcL. En aza indirmek zaman hücreleri, enjeksiyon için önce hücre ölümü azaltmak için süspansiyon geçirirler. </li></ol> 2. Intramiyokardiyal Enjeksiyon <ol><li> Transgenik fareler üzerinde her hücrede yapısal ifade Cre recombinase (B6.C-Tg (CMV-CRE) 1Cgn / J, Jackson Laboratuvarı, Bar Harbor, mühendislik (Phoenix Pharmaceuticals, Inc, St Joseph, MO) izofluran ile anestezinin neden ME). </li><li> Saç kesme makineleri veya kimyasal saç çıkarıcı kullanarak göğüs bölgesinden saç çıkarın. </li><li> - 120 150 mcL bir atım hacmi ile dakikada 130 nefes fare ventilatör 18 gauge kateter (Becton Dickinson &amp; Co, Franklin Göller NJ) ve yeri ile entübe. </li><li> Böylece bir torakotomi üreten dördüncü interkostal alanı boyunca yatay kesi olun. </li><li> Kalp görselleştirme, 1 ml şırınga (Temuro Medical Corporation, Somerset, NJ) ve 28 gauge iğne (Bect kullanarak iki transfekte hücre süspansiyonu 25 mcL enjeksiyonlarıDickinson &amp; Co, Franklin Lakes NJ). Intramiyokardiyal enjeksiyon kolaylığı ve organ aşırı zarar görmesini önlemek için, iğne başı ~ 90 derece bükün. </li><li> Enjeksiyonundan sonra, kaburga ve kas tabakaları kapatmak için emilebilir dikişlerle (örneğin, Vicryl) kullanın. Sütür cilt 4-0 naylon veya ipek kullanılarak kapalı. </li><li> Fare, anestezi ve ekstübe kurtarmak için izin verin. </li><li> Kontrol grupları Cre sadece untransfected hücreleri ve transfekte hücreler (alternatif olarak, Cre fareler sigorta eğilimli transfekte hücreleri) alan vahşi tip fareler aynı konsantrasyonda, Cre fareler orta enjeksiyon uygulanan farelerin içermelidir. </li></ol> 3. Biyofotonik Görüntüleme <ol><li> Görüntüleme, intraperitoneal (IP) 15 mg / mL D-luciferase (Kaliper Yaşam Bilimleri, Hopkinton, MA) fare vücut ağırlığının gram başına 10 mcL enjekte beş ile on beş dakika önce. </li><li> Izofluran üzerinden fareler üzerinde anestezi indüksiyon için% 4 nedenbakım için d 1 -% 2. </li><li> Yüz maskesi 1 idare ile görüntüleme kutusunda yatar Yeri fareler -% 2 izofluran bakım anestezi (Xenogen biyofotonik Görüntüleme Sistemi, Hopkinton, MA). Birkaç fareler aynı anda görüntülü olabilir. Işıklı sinyal kolay karşılaştırma için deneysel fare ile Resim sahte kontrol fare. </li><li> Yaşam Görüntü yazılım (Xenogen) kullanarak, uygun pozlama süresi (genellikle 60 sn, bakınız). Farelerin uyum ve duyarlılık değişiklikleri önlemek için, görüntüleme boyunca alanında tutarlı tutmak için görüntü alan ayarlayın. 4.5 cm konu yüksekliğini ayarlayın. </li><li> Görüş alanı içinde fare ya da fare ile ilgili lüminesans yoğunluğu sinyali Edinme ve değiştirilmemiş görüntü dosyalarını kaydetmek. Süreç görüntüleri bir kontrol ya da unmanipulated fare ile ilgili arka plan sinyali kaldırın. Yoğunluk değerleri yukarıda arka plan hayvan içinde erimiş hücrelere karşılık gelir. Yoğunluk analizi Yaşam Görüntü yazılım (Xenogen) ya da açık kaynak kodlu bir görüntü analiz yazılımı kullanılarak yapılabilir. Typically, ilgi çeken bir bölge (ROI) hayvanlar ve deneyler arasında yoğunluk verileri karşılaştırmak için seçilir. </li></ol> 4. Temsilcisi Sonuçlar Xenogen biyofotonik Görüntüleme Sistemi duyarlılık, yapısal C57/Bl6 fareler (Jackson Laboratory) miyokarda teslim edildi (231-LUC-D3H1, Xenogen) lusiferaz ifade eden bir hücre hattı belirlemek için. Hücreler 1 x konsantrasyonları enjekte edildi 10 6, 1 x 10 3 veya 1 x 10 1 hücre hücre teslim Altı saat sonra, fareler luciferase ile intraperitoneal enjekte Xenogen sistemini kullanarak görüntülü edildi. 1000 hücreleri (2 6 fareler, görüntülü Şekil 2) ile özel bir sinyal tespit, ancak algılama 10,000 hücreleri (görüntülü 6 farelerin 6) ile daha güvenilir olabilir. Daha da önemlisi, bu çalışma aynı zamanda lusiferaz ifade hücreleri ve sinyal yoğunluğu sayısı arasında bir kaba ilişki kurmak için görev yaptı. Kre-ifade farelerin miyokarda teslim edildi. Hücre doğumdan sonra yaklaşık bir hafta, fare ilk kez D-luciferase enjeksiyon olmadan Xenogen sistemi kullanılarak görüntülenmiştir . Enzimatik substrat olmadan, beklenen, hiçbir yoğunluk sinyali (Şekil 3) tespit edildi. Sonra, D-luciferase intraperitoneal ve lusiferaz yoğunluğuna karşılık gelen bir sinyal enjekte edildi ve böylece hücre füzyonu test iki dört farelerde tespit edildi. Bu durumda, çalışma, kalp ve çevresindeki doku değerlendirilmesi için izin sona erdirildi benzer bir sinyal. MSC-çiftli füzyon ürünleri, in vivo olarak korunabilir, en geç bir hafta (Şekil 3) tespit edildi, ancak bir tasavvur olabilir uzun vadeli bir analiz ve bakım, proliferasyon izlemek için daha sık görüntülemeFarelerde füzyon ürünleri d belki de göç. <img alt="Şekil 1" src="/files/ftp_upload/3581/3581fig1.jpg" /> Şekil 1. In Vivo Hücre Fusion Algılama Tekniği şematik arasında füzyon Cre ifade fare hücreleri ve nakledilen hücrelerin meydana gelen bir floxed lusiferaz plazmid ifade, lusiferaz ifade edilmesi durumunda. Lusiferaz Xenogen biyofotonik Görüntüleme Sistemi (19 uyarlanmıştır) kullanarak fare fare enzimatik substrat, D-luciferase, enjekte ve daha sonra görüntüleme ile tespit edilebilir <img alt="Şekil 2" src="/files/ftp_upload/3581/3581fig2.jpg" /> Şekil 2. Tespiti Hassasiyet lusiferaz ifade biyofotonik görüntüleme ile kalp dokusunda hücreler. Yapısal (231-LUC-D3H1, Xenogen) lusiferaz ifade hücre hattı çeşitli toplam hücre sayıları C57/Bl6 farelerin intramiyokardiyal alana teslim edildi. Farelerin Temsilcisi görüntüleri yeniden1 x 10 6, 1 x 10 3 ve 1 x 10 1 hücreleri (soldan sağa) ceiving gösterilmiştir, görüntüleme enjeksiyon yaklaşık 6 saat sonra yapıldı. <img alt="Şekil 3" src="/files/ftp_upload/3581/3581fig3.jpg" /> Şekil 3. Hücre Fusion Endikatif Vivo Lüminesans Niceleme. MKH plazmid LoxP-Stop-LoxP-Lusiferaz ve Kre-ifade farelerin miyokarda teslim ile transfekte. Hücre doğumdan sonra yaklaşık bir hafta ve iki hafta, Cre fareler, hücre füzyonu göstergesidir lüminesans yoğunluğunu ölçmek için Xenogen biyofotonik Görüntüleme Sistemi kullanarak görüntülü. 17 gün hücre doğumdan sonra (A) sham ve fareler 1-4 lüminesans fotoğraf ve yoğunluğu Kaplama (soldan sağa). (B) sham ve fareler 1-4 lüminesans Yoğunluğu (soldan sağa) hücre doğumdan sonra 17 gün. Ilgi bölgeye enjeksiyon yerinde ve yoğunluğu l uygun seçildi (sarı)evels ImageJ (ücretsiz kaynak) yazılımı 20 kullanılarak belirlenmiştir. (C) lüminesans yoğunluğu tüm deneysel koşullar altında, aynı bölgede sahte fare faiz normalize edildi. Bir hafta, 3 ve 4 fareler, bir fare hücre ve nakledilen MSC spontan füzyon düşündüren pozitif lüminesans sinyal gösterdi. Fare 3 sinyali iki hafta devam etti. Fare 3 karşılık gelen sinyal organ belirli bir yeri belirlemek için, göğüs boşluğu maruz kalan ve birincil organları eksize görüntülendi. (D), fotoğraf ve fare 3 lüminesans yoğunluğu Yerleşimi. Not yerelleştirme ince bağırsakta yoğunluğu sinyali. (E) Yoğunluk fare 3 lüminesans. (F), fotoğraf ve sahte fare lüminesans yoğunluğu Yerleşimi. (G) Yoğunluk sahte fare lüminesans.

<ol>
	<li>Bernirschke, K. K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology of the Human Placenta. , (2000).</li><li>Hoshina, M., Boothby, M., Boime, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+localization+of+chorionic+gonadotropin+alpha+and+placental+lactogen+mRNAs+during+development+of+the+human+placenta.">Cytological localization of chorionic gonadotropin alpha and placental lactogen mRNAs during development of the human placenta.</a> J. Cell. Biol. 93, 190-198 (1982).</li><li>Johansen, M., Redman, C. W., Wilkins, T., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophoblast+deportation+in+human+pregnancy--its+relevance+for+pre-eclampsia.">Trophoblast deportation in human pregnancy--its relevance for pre-eclampsia.</a> Placenta. 20, 531-539 (1999).</li><li>Redman, C. W., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+debris,+oxidative+stress+and+pre-eclampsia.">Placental debris, oxidative stress and pre-eclampsia.</a> Placenta. 21, 597-602 (2000).</li><li>Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+fusion+of+cells+between+species+yields+transdifferentiation+and+retroviral+transfer+in+vivo.">Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo.</a> FASEB. J. 18, 548-550 (2004).</li><li>Nygren, 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=Bone+marrow-derived+hematopoietic+cells+generate+cardiomyocytes+at+a+low+frequency+through+cell+fusion,+but+not+transdifferentiation.">Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.</a> Nat. Med. 10, 494-501 (2004).</li><li>Nygren, 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=Myeloid+and+lymphoid+contribution+to+non-haematopoietic+lineages+through+irradiation-induced+heterotypic+cell+fusion.">Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion.</a> Nat. Cell. Biol. 10, 584-592 (2008).</li><li>Kutschka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+human+BCL-2+transgene+expression+attenuates+early+donor+cell+death+after+cardiomyoblast+transplantation+into+ischemic+rat+hearts.">Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts.</a> Circulation. 114, I174-I180 (2006).</li><li>Min, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+of+cord+blood+derived+mesenchymal+stem+cell+transplantation+into+rat+myocardium.">In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium.</a> Ann. Nucl. Med. 20, 165-170 (2006).</li><li>Malstrom, S. E., Tornavaca, O., Meseguer, A., Purchio, A. F., West, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+characterization+and+hormonal+regulation+of+kidney+androgen-regulated+protein+(Kap)-luciferase+transgenic+mice.">The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice.</a> Toxicol. Sci. 79, 266-277 (2004).</li><li>Weir, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophotonic+imaging+in+HO-1.luc+transgenic+mice:+real-time+demonstration+of+gender-specific+chloroform+induced+renal+toxicity.">Biophotonic imaging in HO-1.luc transgenic mice: real-time demonstration of gender-specific chloroform induced renal toxicity.</a> Mutat. Res. 574, 67-75 (2005).</li><li>Rajashekara, G., Glover, D. A., Banai, M., O'Callaghan, D., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+bioluminescent+Brucella+melitensis+mutants+GR019+(virB4),+GR024+(galE),+and+GR026+(BMEI1090-BMEI1091)+confer+protection+in+mice.">Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.</a> Infect. Immun. 74, 2925-2936 (1090).</li><li>Kadurugamuwa, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biophotonic+imaging+for+monitoring+of+catheter-associated+urinary+tract+infections+and+therapy+in+mice.">Noninvasive biophotonic imaging for monitoring of catheter-associated urinary tract infections and therapy in mice.</a> Infect. Immun. 73, 3878-3887 (2005).</li><li>Ryan, P. L., Youngblood, R. C., Harvill, J., Willard, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photonic+monitoring+in+real+time+of+vascular+endothelial+growth+factor+receptor+2+gene+expression+under+relaxin-induced+conditions+in+a+novel+murine+wound+model.">Photonic monitoring in real time of vascular endothelial growth factor receptor 2 gene expression under relaxin-induced conditions in a novel murine wound model.</a> Ann. N.Y. Acad. Sci. 1041, 398-414 (2005).</li><li>Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+imaging+of+GFAP+expression+after+neuronal+damage+in+mice.">Non-invasive imaging of GFAP expression after neuronal damage in mice.</a> Neurosci. Lett. 367, 210-212 (2004).</li><li>Noiseux, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+overexpressing+Akt+dramatically+repair+infarcted+myocardium+and+improve+cardiac+function+despite+infrequent+cellular+fusion+or+differentiation.">Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation.</a> Mol. Ther. 14, 840-850 (2006).</li><li>Ajiki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+tissue+transplantation+in+rats:+fusion+of+donor+muscle+to+the+recipient+site.">Composite tissue transplantation in rats: fusion of donor muscle to the recipient site.</a> Transplant Proc. 37, 208-209 (2005).</li><li>Trivedi, P., Hematti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+immunological+characterization+of+mesenchymal+stromal+cells+from+human+embryonic+stem+cells.">Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells.</a> Exp. Hematol. 36, 350-359 (2008).</li><li>Ogle, B. M., Cascalho, M., Platt, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+cell+fusion.">Biological implications of cell fusion.</a> Nat. Rev. Mol. Cell. Biol. 6, 567-575 (2005).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+microscopy.">ImageJ for microscopy.</a> BioTechniques. 43, 25-30 (2007).</li></ol>

Çıkar çatışması ilan etti.

Bu yöntem küçük hayvanlar da dahil olmak üzere, canlılar, ilk kez izin verir hücre füzyonu ayrık tanımlama ve temporal analiz tarif. Bu yaklaşım daha sonraki biyofotonik görüntü analizi ile Cre-LoxP rekombinasyon birleştirir. Sadece hücre-hücre füzyonu izlemeye müsait bir yaklaşım, aynı zamanda virüs hücre füzyonu ve benzeri viral enfeksiyonlar izlemek için faydalı olabilir. Görüntü analizi hızlı ve görüntü aynı anda birden fazla küçük hayvanlar için mümkündür. Füzyon...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Neon Transfeksiyon Sistemi </td><td> Invitrogen, Carlsbad, CA </td><td> MPK5000 </td><td></td></tr><tr><td> Neon 100 mcL Kiti </td><td> Invitrogen, Carlsbad, CA </td><td> MPK10025 </td><td> R ve E Tampon içerir. </td></tr><tr><td> a-MEM toz </td><td> Invitrogen, Carlsbad, CA </td><td> 12000-022 </td><td></td></tr><tr><td> Fetal sığır serumu (FBS) </td><td> Hyclone, Logan, UT </td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C-Tg (CMV-CRE) 1Cgn / J </td><td> Jackson Laboratory, Bar Harbor, ME </td><td> 006054 </td><td></td></tr><tr><td> 10X tripsin </td><td> Fisher Scientific, Forest Lawn, NJ </td&gt; <td> MT-25-054-Cl </td><td></td></tr><tr><td> L-Glutamine </td><td> Fisher Scientific, Forest Lawn, NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D-luciferase </td><td> Kaliper Yaşam Bilimleri, Hopkinton, MA </td><td> 122796 </td><td></td></tr><tr><td> Xenogen biyofotonik Görüntüleme Sistemi </td><td> Kaliper Yaşam Bilimleri, Hopkinton, MA </td><td> IVIS Spektrum </td><td></td></tr><tr><td> Sodyum Biocarbonate </td><td> Sigma Aldrich, St Louis, MO </td><td> S6014-500G </td><td></td></tr><tr><td> Sigara esansiyel amino asitler </td><td> Invitrogen, Carlsbad, CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

Sigorta için aynı tipte iki veya daha fazla hücre yeteneği, iskelet, kas, kemik ve plasenta da dahil olmak üzere pek çok kompleks organ, form, evrim boyunca metazoans kullanılmıştır. Çağdaş çalışmalar aynı tip hücrelerin füzyonu gelişmiş fonksiyonu bahşeder göstermektedir. Örneğin, plasenta sigorta trofoblast hücreleri syncytiotrophoblast formu ne zaman, syncytiotrophoblast daha iyi bir halkaya yapışmamış trofoblast 1-4 daha maternal-fetal bariyer boyunca besin ve hormonlar taşımak mümkün. Daha yeni çalışmalarda farklı tipte hücrelerin füzyonu, hücre kaderini doğrudan göstermektedir. Füzyon &quot;reversion&quot; veya hücre kaderinin değiştirilmesi kez hücre kültürü sistemleri ile sınırlı olduğu düşünülüyordu. Ama kök hücre nakli gelişi bize keşfine yol açtı ve, kök hücre in vivo somatik hücre ile sigorta ve kök hücre farklılaşması füzyon 5-7 kolaylaştırır . Böylece, hücre füzyonu düzenlenmiş bir süreç cböylece, hücre yaşamını ve farklılaşmasını teşvik apable ve gelişme, doku onarımı ve hatta hastalığın patogenezinde için merkezi bir önemi olabilir. Uygun teknoloji eksikliği) 1, hücre füzyon çalışma sınırlandırılması doğru zaman içinde) izlemek füzyon ürünleri füzyon ürünleri belirlemek ve 2. Burada füzyon üzerine biyoparlaklık indüksiyon (Şekil 1) ile iki kısıtlamalarını ortadan kaldırmak için yeni bir yaklaşım mevcut. Biyoparlaklık in vivo 8-15 yüksek hassasiyet ile tespit edilebilir bir yapı ateşböceği lusiferaz kodlama kullanır (Photinus pyralis) gen bitişik yerleştirilen bir stop kodon LoxP dizileri ile çevrili. Cre recombinase protein ifade hücreleri ile bu gen sigorta ifade hücreleri LoxP siteleri bölünmüş ve stop sinyali lusiferaz transkripsiyonunu izin eksize edilir. sinyali induci olduğundanble, yanlış pozitif sinyallerin sıklığı çok düşüktür. Cre / LoxP sistemi 16, 17 kullanan mevcut yöntemlerden farklı olarak, bir &quot;canlı&quot; bir saptama sinyali dahil ve böylece ilk defa hücre füzyon kinetiği, in vivo olarak takip etme olanağı göze. Yaklaşım göstermek için, yayg Cre recombinase ifade fareler, bir floxed stop kodon lusiferaz downstream ifade etmek için bir yapı ile transfekte kök hücre alıcıları olarak görev yaptı. Kök hücreler intramiyokardiyal enjeksiyon yoluyla nakledilen ve nakli intravital varlığını izlemek için görüntü analizi yapıldı zaman içinde kalp ve çevresindeki dokularda füzyon ürünleri. Bu yaklaşım, geliştirme, hastalık veya yetişkin doku onarımı herhangi bir aşamasında, herhangi bir doku tipi hücre füzyon analiz adapte olabilir.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

, Zaman içinde canlı organizmaların hücre füzyon izlemek için bir yöntem açıklanmıştır. Yaklaşım kullanır Cre<em&gt; LoxP</em&gt; Rekombinasyon hücre füzyonu üzerine lusiferaz ifade ikna etmek için. Periferik dokularda ~ 1.000 hücrelerinin algılama duyarlılığı ile biyofotonik görüntüleme sistemleri kullanılarak canlı organizmalarda üretilir Işıklı sinyal tespit edilebilir.

Hücre Fusion Tespit Cre-ETOX P Rekombinasyon Yaklaşım In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

a-cre-lox-p-recombination-approach-for-detection-cell-fusion

Research

JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

18.7K Görüntüleme.  University of Wisconsin-Madison. , Zaman içinde canlı organizmaların hücre füzyon izlemek için bir yöntem açıklanmıştır. Yaklaşım kullanır Cre LoxP Rekombinasyon hücre füzyonu üzerine lusiferaz ifade ikna etmek için. Periferik dokularda ~ 1.000 hücrelerinin algılama duyarlılığı ile biyofotonik görüntüleme sistemleri kullanılarak canlı organizmalarda üretilir Işıklı sinyal tespit edilebilir.

Video: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biyomoleküler Algılama istihdam İnterferometrik Yansıtma Görüntüleme Sensörü (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Biyomühendislik

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Nükleik Asitler, Proteinler ve Küçük Moleküller Reagentless Algılama Elektrokimyasal DNA Biyosensörler Fabrikasyon

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Protein ve hücre Etrafının İki Boyutlu Desenli Yüzeyler Oluşturma

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Için basit Mikroakışkan Cihazlar<em&gt; In vivo</emVe&gt; Görüntüleme<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Ve Zebra balığı

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Mikroenjeksiyon Yara Deneyi ve<em&gt; In vivo</em&gt; Epidermal lokalizasyonu Tepki Gazeteciler Yara<em&gt; Drosophila</em&gt; Embriyolar.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A Tam Cilt Hata Modeli Biyomateryallerin vaskülarizasyonunu değerlendirin<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Tek Hücre Salgıları uzay-zamansal Görüntüleme için bir etiket içermeyen Tekniği

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

için içi boş fiber Biyoreaktörler<em&gt; İn Vivo</em&gt; Benzeri Memeli Doku Kültürü

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D Etkileşimleri Çalışmaları 3D Kollajen jeller ve Mikrokanallarda Hazırlanması<em&gt; İn Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Birden çok nükleik asit kapiller bir dizi görsel algılama

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...