Biz tüm Caldwell Lab üyeleri işbirliği ruhu kabul etmek istiyoruz. Bachmann-Strauss Distoni ve Parkinson Vakfı, Birleşik Parkinson Vakfı, Amerikan Parkinson Hastalığı Derneği, Parkinson Hastalığı Derneği Alabama, Michael J. Fox Parkinson Araştırma Vakfı ve Lisans Araştırma laboratuarında Hareket bozuklukları araştırma tarafından desteklenmektedir.

İfade Plazmid yapı İki plazmid gereklidir: doku özel ilgi gen ekspresyonu ve seçilebilir dönüşüm belirteci olarak ikinci bir biri. Deneysel Plazmid <ol><li> Örneğin faiz doku / hücre tipi, bir sinir ya da kas-özel organizatörü olarak ifade organizatörü seçin. Aynı doku içinde birden fazla genin ifade etmek için ise, Gateway sisteminin (Invitrogen) plazmid bir yararlanıcı kaset kullanılabilir. Bu yaklaşım, recombinational klonlama 4 organizatörü faiz downstream herhangi bir gen ekleme sağlar. Genellikle 2-5 kb upstream dizilerinin doğru ve doğru ifade etmek için yeterli. </li><li> CDNA ilgi genin iki şekilde klonlanmış olabilir: <ol><li> Restriksiyon enzimlerini kullanarak Konvansiyonel klonlama. </li><li> Gateway klonlama için, PCR Ağ Geçidi att B recombinational dizisi içeren primerler kullanılarak amplifiye. Amplifiye cDNA ilk giriş vektörü oluşturmak için donör vektör pDONR201 recombined ve sonra E. dönüştü coli suşu DH5a. Başarılı rekombinantlar ve DNA mini hazırlık izolasyon seçimi takiben, giriş vektörü ifade plazmid yaratmak için organizatörü içeren hedef vektör recombined. Recombinational klonlama Ayrıntılar Invitrogen Ağ Geçidi manuel veya Caldwell ve ark mevcuttur. 5. </li></ol></li></ol> Plazmid Seçilebilir Marker Transgenik işaret plazmid örnekleri, rol-6 ya da vücut duvarı kas organizatörü unc-54 floresan protein ekspresyonu sürücü kullanımı sayılabilir. Bu işaretleyici plazmidler genellikle istek üzerine araştırma toplum içinde mevcuttur. C. elegans, Mikroenjeksiyon Hazırlık <ol><li> Enjekte edilecek olan iki plazmid DNA izolasyonu gerçekleştirin. Asitli ve 50 ng / ul her 1:1 oranında karıştırın. </li><li> Agar pedleri hazırlayın: <ol><li> Eriyene kadar ısı veya mikrodalga% 2 agaroz su çözümü olun. </li><li> Bir masa üstü birkaç 22 x 50 mm kapak camları yola çıktı. </li><li> Pasteur pipeti kullanarak, bir kapak cam erimiş agaroz bir damla koyun ve hemen ilk 90 açıyla açılan üzerinden ikinci bir cam kapak yerleştirin. Diğer birçok kapak camları için bu işlemi tekrarlayın. Agaroz basık disk çapı 15-20 mm arasında olmalıdır. </li><li> Agaroz (Bu dakikanızı alır) katılaşmış sonra, kapak camını çıkarın ve pedi tamamen kurumaya izin (bir gecede birkaç saat). </li><li> Cam bir kapakla kutusunda yastıkları, oda sıcaklığında saklayın. </li></ol></li><li> DNA çözümü için iğne yükleyiciler olun: İğne yükleyiciler ortasında açık ateşte ısıtılır ve boyları yaklaşık iki katı hızla çekti 100 ul kılcal tüpler oluşturulur. Kılcal tüp soğutulduktan sonra iki yarısı ayrı tersledi. 10-20 enjeksiyon için iğne yükleyiciler Stoğu. Dik bir mikrosantrifüj tüp rafta saklayın. Noktalarda kendini Impale olarak dikkatli kullanın. </li><li> Mikroenjeksiyon iğne yapın: Bir iç cam elyaf içeren özel bir kılcal tüpten iğne yapılır; bu filament DNA çözümü için bir fitil olarak hizmet vermektedir. Bu iğne çektirmenin makinede çekilir. Biz bir ısı ayarı ile 24.8 Narishige PP-830 çektirmesi kullanın. Bir seferde birkaç iğne çekilir ve küçük bir plastik kutu içinde saklanır. Birden fazla iğne kolayca modelleme kil, uzun süreli depolama için bir şerit üzerinde &quot;monte&quot; olabilir. </li><li> İğne ucu &quot;kesiciler&quot; Mikroenjeksiyon iğne ucu kadar ince aslında sonu kapalı olduğunu çekilir ve küçük kapak cam kırıkları ile açık kırık olmalıdır. Bu bir kağıt havlu, 18 x 18 mm kapak cam ambalaj ve birkaç parçaya kırar kadar hafifçe basınç uygulayarak tarafından hazırlanır. Shard yararlı bir boyutu yaklaşık 3 x 4 mm. </li><li> Vahşi tip (N2) solucanlar standart prosedürler kullanarak 6 enjeksiyon için erken yetişkinlik evresi için büyütün. Yaklaşık 100 hazırlayın düzgün solucanlar sahnelenen / inşa enjekte. </li></ol> Prosedür <ol><li> Mikroenjeksiyon iğne kol ve sahibine bağlı olan bir helyum tankı ana vanayı açın. Gaz basıncı 35 psi getirerek, çizgi girmek için izin vermek için regülatör vanasını kapatın. Ayak pedalı ile gaz açığa çıkabilir dikkat edin. </li><li> Bir iğne yükleyici, standart bir ağız pipetter cam kapiller tüplerin kaplar içinde bulunan tüp içine yerleştirin ve plazmid karışımı (~ 1 ul) küçük bir miktar yukarı çekmek. </li><li> Iğne yükleyici bir enjeksiyon iğnesi arka uç dikkatlice yerleştirilir ve hafifçe uzunluğu boyunca tüm yol takılıiğne kadar direnç hissedilir. Hafifçe üfleyerek DNA çözüm kovduktan sonra yükleyici çıkarın. </li><li> Küçük kauçuk conta içinde yerleştirmek için dikkatli olmak, mikroenjeksiyon koluna iğne takın. </li><li> Agar bir ped bir kenarı cam kapak iğne kırma shard yerleştirin. Halokarbon yağ ile, shard, agar pad gibi tüm yüzeyi örtün. Ters bir mikroskop sahnede agar yastık yerleştirin. </li><li> 4X objektif ve parlak bir alan aydınlatma kullanarak görüş alanının merkezinde iğne pozisyonu, ama henüz yağa düşürmez. Ayrıca, merkezde kapak cam shard iç kenarı konumu, o zaman odaklanmak (hala 4X objektif kullanarak). Cam kapak shard aynı odak düzlem haline getirerek, yağ içine iğne indirin. 40X objektif geçiş büyütme kaldırın. Shard kenarı üzerinde odaklanılması ve gerekirse iğne ucu yeniden konumlandırmak. </li><li> Ayak pedalı ile gaz kısa bir darbe uygulayarak, aynı zamanda da bir iğne ucu kadar açmak için, yavaşça, kapak cam Shard kenarına iğne dürtmek. Sıvı küçük damlacıklar iğne sonunda kaçmak iğne yeterince açık kırılmış olacaktır. Hayvanları öldürecek gibi çok büyük bir açılış nedeniyle Aşırı sıvı akış, arzu edilen değildir. </li><li> Iğne kadar yükselterek aşamasından agar pedini, ancak X ya da Y ekseni konumunu değiştirmez. Aşamada iğnenin altından dışarı doğru kaydırın, agar pad kaldırmak ve diseksiyon mikroskobu üzerine yerleştirin. Petrol yüzeyinin altında bir solucan, ped üzerine aktarın. Eğer solucan wriggles, yavaşça felç temas agar pad kadar. Nemli solucan kuru agaroz uymalıdır. </li><li> Enjeksiyon <ol><li> Hızla, sahneye agar pad yerine geri görüş alanı solucan merkezleme. </li><li> Solucan gibi odak aynı düzlem içine iğne indirin. </li><li> Hoffman Işık (kontrast filtre türü) filtreleri geçin. Bu solucanın morfolojik ayrıntı görünür olmasını sağlar. Nomarski / DIC optik ters kapsamı üzerinde de çalışacak. 40X objektif kullanarak, mikrop çekirdeğinin &quot;petek&quot; desen altında solucan gonad &quot;grenli&quot; sinsityal merkezi, (solucan iğne bakan tarafında konumlandırılmış hangisi gonad seçin) odaklanmak. </li><li> İnce ayar iğne ucu (gonad &quot;grenleşme&quot; olarak aynı düzlemde) odak kadar pozisyonu. </li><li> Gonad merkezi haline hafifçe iğne takın ve DNA ile ilgili bir ya da iki damlacık gonad kol sınırdışı gaz basıncı kısa bir darbe uygulayın. Distal gonad boyunca sıvı yayılmış bir dalga dikkat etmelisiniz. Oosit üretim kapatabilirsiniz gonad kol proksimal viraj içine çok fazla sıvı akabilir yılından bu yana daha fazla sıvı, daha iyi kabul etmeyin. Eğer mümkünse, solucanın pozisyonuna bağlı olarak, aynı şekilde diğer gonad kol enjekte edilir. </li><li> Kolaylıkla birbirinden ayrılacak gibi, bir solucan enjekte ederken hızlı çalışması için önemlidir. Ikinci gonad kol enjekte kararı iğne ve solucan yeniden pozisyonu ne kadar hızlı bağlıdır. Tüm süreci, ideal olarak 1-2 dakika daha az almalıdır. </li></ol></li><li> Aşamasından cam kapak çıkarın ve diseksiyon kapsamı üzerine yerleştirin. Doğrudan enjekte solucan üzerine M9 tampon 1μl (22mm KH 2 PO 4, 42mm, Na 2 HPO 4, 85mm NaCl, 1mm MgSO 4) (Bu Halokarbon petrol yüzeyinin altında pipet daldırarak yol açabilir) uygulayın. Tampon solucan rehidrate; sonra agaroz ped yüzeyi yüzer. Bir solucan almak kullanarak normal bir plaka solucan aktarın. Agaroz ped, tampon ve solucanlar artık uymak çok doymuş hale kadar birkaç kez daha tekrar edilebilir. </li></ol> Transgenik seçimi <ol><li> Enjekte solucanlar, P 0 nesil, bireysel taze, bakteriyel numaralı seribaşı levhalar (60 mm) aktarılır ve üremek için izin verilir. Tipik olarak, enjekte edilen hayvanların yavrular ortaya çıkıncaya kadar 2-3 gün süreyle 20 ° C&#39;de inkübe edilir. </li><li> F 1 yavrular floresan stereomikroskopta kullanarak transgenik marker (örneğin floresans) kanıt görülmektedir. Her floresan, taşıyıcı F 1, hayvan ayrı ayrı (her F 1 yavrulara ayrı bir hat kabul edilir) yeni bir plaka üzerine klonlanmış olduğunu. F 1 çift cinsiyetli, üremek için izin verilir . Yine, yavrular ortaya çıkıncaya kadar bu genellikle 20 ° C de 2-3 gün için yapılır. </li><li> F 2 nesil transgenik hayvanlar için de görülmektedir. F 2 hayvan miras ve transgenik dizi ifade &quot;istikrarlı&quot; bir çizgi olarak kabul edilir . NOT: F 1 styaşı birçok transgenik hayvanlar olabilir, ama onlar kararlı değildir. F 1 transgenik hayvanlar çoğunluğu F 2 istikrarlı hatları içine yayılır olmayacaktır. NOT: gen kopya sayısı değişkenliği kontrol etmek için, en az enjekte inşa başına üç bağımsız sabit hatları oluşturur . </li><li> Her bağımsız istikrarlı bir çizgi solucanlar ayrı bir çizgi olarak muhafaza edilmelidir. Her satır, her nesil yeni bir plaka birkaç transgenik hayvanlar transfer tarafından yayılan olabilir; seçilebilir marker floresan ise bu floresan stereomikroskopta kullanarak yapılmalıdır. </li></ol>

<ol>
	<li>Stinchcomb, D. T., Shaw, J. E., Carr, S. H., Hirsh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrachromosomal+DNA+transformation+of+Caenorhabditis+elegans.">Extrachromosomal DNA transformation of Caenorhabditis elegans.</a> Mol. Cell. Biol. 5, 3484-3496 (1985).</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>Kelly, W. G., Xu, S., Montgomery, M. K., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+requirements+for+somatic+and+germline+expression+of+a+generally+expressed+Caenorhabditis+elegans+gene.">Distinct requirements for somatic and germline expression of a generally expressed Caenorhabditis elegans gene.</a> Genetics. 146, 227-238 (1997).</li><li>Cao, S., Gelwix, C. C., Caldwell, K. A., Caldwell, 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=Torsin-mediated+neuroprotection+from+cellular+stresses+to+dopaminergic+neurons+of+C.+elegans.">Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans.</a> J Neurosci. 25, 3801-3812 (2005).</li><li>Caldwell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Integrated Genomics: A Discovery-Based Laboratory Course. , (2006).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

Bu işlemi yaparken, hatırlamak önemlidir: - Gonad merkezi içine doğrudan enjekte - Çok fazla sıvı enjekte - Kuruma önlemek için hızlı bir şekilde çalışır. Çok büyük kırık gibi iğne ile ilgili sorunlar yaşarsanız, bu daha az ideal bir iğne ile enjekte etmeye çalışıyor yerine, taze bir iğne yeniden en iyisidir. Transgenik solucanlar nesil gibi birçok uygulama vardır: - Mutant genlerin belirlenmesi, dönüşüm kurtarma yöntemi denir - Kesilmiş proteinleri if...

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<td>Agarose</td>
<td>Ultrapure</td>
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<td>elfatochem, France</td>
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<td>Coverglass 18x18 mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
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<td>Coverglass 20x30 mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
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generation of stable transgenic c. elegans using microinjection

Transgenik Caenorhabditis elegans gonad 1 DNA plazmid çözüm mikroenjeksiyon ile kolayca oluşturulabilir. Plazmid DNA, stabil bir şekilde olsa da gerçek kromozom 2 olarak aynı verimlilikle değil, miras alınır ekstrakromozomal concatamers oluşturmak için yeniden düzenler . Diseksiyon mikroskobu altında transgenik hayvanlar seçimi izin vermek, ilgi bir gen rol-6 ya da GFP gibi bariz bir fenotipik işaretleyici ile ortak enjekte edilir. Eksojen gen hücresel yerelleştirme çalışmaları için ana organizatörü ifade edilebilir. Alternatif olarak, transgen, bu belirli bir hücre ya da doku gen ürünü rolünü değerlendirmek için farklı bir doku-spesifik organizatörü tarafından sürülebilir. Bu teknikte verimli C. elegans germline ya da erken embriyo 3 hariç bütün dokularda gen ekspresyonu sürücüler. Transgenik hayvanlar oluşturulması, deneysel paradigmalar bir dizi için yaygın olarak kullanılmaktadır. Bu video transgenik solucanları oluşturmak için mikroenjeksiyon işlemi göstermektedir. Ayrıca, istikrarlı transgenik C. elegans hatları seçimi ve bakım açıklanmıştır.

Watch this Scientific Journal Video about Generation of Stable Transgenic C. elegans Using Microinjection at JoVE.com

Generation of Stable Transgenic C. elegans Using Microinjection

Bu video transgenik hayvanlar oluşturmak için C. elegans gonad içine mikroenjeksiyon tekniği göstermektedir.

Mikroenjeksiyon Kararlı Transgenik C. elegans Üretimi

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

generation-of-stable-transgenic-c-elegans-using-microinjection

Research

JoVE Journal

Biology

52.7K Görüntüleme.  University of Alabama. Bu video transgenik hayvanlar oluşturmak için C. elegans gonad içine mikroenjeksiyon tekniği göstermektedir.

Video: Generation of Stable Transgenic C. elegans Using Microinjection

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Transgenik Sivrisinekler Edinme A. aegypti Embriyolar Mikroenjeksiyon

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Biyoloji

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Transgenik, Üretimi C. elegans Biyolistik Dönüşüm

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Görüntüleme C. elegans Embriyolar Epifluorescent Mikroskop ve Açık Kaynak Yazılım

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

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 ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Genetik İfade Profilleri Belirlenmesi C. elegans Mikroarray ve Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans Kemotaksis Deneyi

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Antikor boyamasında<em&gt; C. Elegans</em&gt; &quot;Freeze-Cracking kullanma&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Üzerinde Bakterilerin Etkilerinin Ölçme<em&gt; C. Elegans</emBir Yumurta Tutma Testi Kullanma&gt; Davranış

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

Transgenik Üretimi<em&gt; Hydra</em&gt;: Mikroenjeksiyon yolu ile embriyo

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Metazoan Model Organizma kullanma Serim ve toksisitesi prion-benzeri Proteinler incelenmesi<em&gt; ° C. elegans</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...