Este trabalho foi financiado pela NSF conceder IOB # 0615996.

Parte 1: Preparando o crescimento da mídia nematóide (NGM) placas de Petri Nematóides experimentais como C. elegans e P. pacificus são normalmente cultivadas em laboratório em 6 placas de Petri contendo meio centímetro Nematóides do Crescimento (NGM) 1. Worms são mantidos a 20 ° C, mas pode ser incubado a uma gama de temperaturas (entre 4 ° C - 25 ° C), dependendo da espécie de nematóides e design experimental. Para preparar pratos com NGM, standard técnicas estéreis devem ser utilizados para evitar a contaminação por fungos e bactérias. Segue-se o protocolo para preparar placas NGM: <ol><li> Pesar 15 g de ágar, 2,4 g NaCl, 2 g Triptona, e 2,72 g KH 2 PO 4 em 2 L frasco cônico, fazer volume até 1 L com autoclave, água e deixe-o esfriar até 60 ° C em banho-maria. </li><li> Adicione 0,8 ml cada, de 1 M CaCl 2, colesterol (5 mg / ml em etanol) e 1 M MgSO 4. Para evitar a contaminação por fungos e bactérias, 1 estreptomicina ml (100 mg / ml) e nistatina 1ml (10 mg / ml) podem ser adicionados a cada litro de meio. </li><li> Usando uma máquina Unispense e manter condições estéreis, despeje a mídia nas placas desejado. As placas devem ser preenchidos para cerca de 2 / 3 rd completo. Um padrão de 60 mm de diâmetro placa requer cerca de 10 ml dos meios de comunicação. Não mova as placas até que o ágar esteja completamente gelified, caso contrário haverá uma superfície irregular de agar, o que torna difícil se concentrar worms na estereomicroscópio. </li><li> Depois de secas, as placas podem ser semeadas imediatamente ou armazenados a 4 ° C até a semeadura com bactérias. </li></ol> Parte 2: Preparando OP50 bactérias e semeadura placas NGM Petri P. pacificus é cultivado em laboratório, alimentando a tensão OP50 E.coli. OP50 tem um crescimento limitado em placas NGM, formando um gramado fino que facilita a visualização dos worms 1. A crescer uma cultura de OP50, use o caldo LB: <ol><li> Adicionar 5 g bactotryptone, 2,5 g de extrato de levedura e 2,5 g de NaCl para um frasco de 500 ml tampa de rosca, faça aumentar o volume para 500 ml com água destilada e autoclave. </li><li> Inocular o caldo com uma única colônia de OP50 da placa de cultura em condições estéreis e deixá-lo crescer overnight a 37 ° C. </li><li> LB-OP50 está pronto para ser usado e pode ser armazenado a 4 ° C durante vários meses, se a esterilidade é mantida. Sempre que as placas precisam ser semeados, despeje uma pequena quantidade do caldo em um copo pequeno estéril e isso vai ajudar a evitar a contaminação da cultura estoque. </li><li> Para placas de semente, dispensar 100-120 mL da OP50 para a 60 mm de diâmetro NGM placa de Petri e agite para espalhá-lo. </li><li> Incubar estas placas semeadas durante a noite a temperatura ambiente para o relvado a crescer. Estas placas semeadas agora podem ser armazenados a 4 ° C por até 3 semanas. Armazenando-os em uma posição invertida ajuda a evitar uma secagem rápida. </li></ol> Parte 3: Fazendo uma picareta Nesta seção descrevemos como fazer uma escolha para vermes. A picareta é usado para transferir worms de um local para outro. A escolha é feita de 30 a 90% fio de platina irídio calibre 10%, embora alguns pesquisadores podem preferir composições de metal ligeiramente diferente: <ol><li> Cortar cerca de 3-4 cm de fio, coloque 0,5 cm de la dentro a ponta de uma pipeta Pasteur (cuja ponta foi quebrada a um menor comprimento), e depois selá-la no vidro sobre um bico de Bunsen. O comprimento do fio saindo do vidro é de cerca de 3-3,5 cm, mas pode variar de acordo com as preferências individuais. </li><li> Achatar a extremidade do fio que está saindo com um martelo ou alicate. Em seguida, dobre para cima porção achatada para formar uma colher. </li><li> Suavizar as bordas afiadas da pick com uma lixa para evitar danos ao worm ou o ágar. </li></ol> Parte 4: worms Picking <ol><li> Para escolher worms, esterilizar o fio de platina em uma chama e arraste a ponta achatada da pick ao longo do gramado bacteriana para revestir a ponta com espessura bactérias pegajoso, tomando cuidado para não perfurar ou danificar a superfície do ágar. </li><li> Usando o estereomicroscópio, muito levemente a ponta da escova pegajosa contra os vermes worm / para ser escolhido até o worm varas para as bactérias na escolha. </li><li> Uma vez que o worm está na ponta da picareta, transferi-lo imediatamente para a nova placa tocando ou deslizando a ponta da picareta contra o gramado bacteriana da placa de novo, eo bicho deve rastrear off. Cuidados devem ser tomados para não danificar a superfície do agar na placa mais os vermes rastejam nos orifícios que torna difícil recuperá-los. Se o worm permanece na escolha por muito tempo, ele pode desidratar. </li></ol> Parte 5: EMS mutagênese Etílico metano sulfonato (EMS) induz a um amplo espectro de mutações no genoma do 2. Mutantes podem ser identificados por defeitos como a postura de ovos, defeitos muscularess, a determinação do sexo, formação Dauer, comportamento e formação das gônadas. O protocolo para EMS mutagênese é semelhante ao descrito para C. elegans 1. <ol><li> Prepare 500 ml (ou mais) de NaOH 1N. </li><li> Tome 4-5, 6 cm de diâmetro placas de Petri cheia de vermes, contendo algumas centenas de worms na terceira larval juvenil (J3) palco e lavar os worms fora de usar 2-3 ml de M9 estéril por placa. </li><li> Recolher todos os worms em uma estéril, descartável tubo de centrifugação de 15 ml. Centrifugar a 1500 xg por 5-7 minutos ou até que os vermes formam uma pelota. </li><li> Aspirar o líquido e usar 2 ml de M9 para re-suspender os vermes. Adicionar 20 mL EMS para um tubo contendo 2 ml de M9 e agite para dissolvê-lo. Em seguida, adicione esta solução EMS ao 2ml de suspensão worm e homogeneizar suavemente. A concentração final da EMS será 47 mM. Atenção: A EMS é um mutagênico muito forte e deve ser tratado em uma capela. Todos os materiais (luvas, pipetas, dicas) que entram em contato com este agente mutagénico deve ser tratada com NaOH 1N para inativo da EMS. Usar luvas duplo durante todo o procedimento mutagênese EMS. Descarte os resíduos perigosos de acordo com as diretrizes da instituição. </li><li> Coloque o tubo de centrifugação e cobrir a tampa com parafilme; colocá-lo horizontalmente no extractor de fumo e deixar as minhocas incubar por 3,5 horas. O tubo também pode ser colocado em um rocker de baixa velocidade. </li><li> Vire o tubo e cobrir a tampa com parafilme; lugar na posição vertical e incubar até que o dissipador worms. </li><li> Aspirar o sobrenadante e descartá-lo em um copo / frasco contendo NaOH 1N. Isto irá desativar o EMS. </li><li> Adicionar 5 ml M9 aos vermes, inverter o tubo 25-30 vezes para lavar os worms, centrifugar a 1500 xg por 5-7 minutos ou até que os vermes formam uma pelota. Aspirar o sobrenadante e descartar no copo NaOH 1N. </li><li> Repita o passo de lavagem, pelo menos, três vezes. </li><li> Após a lavagem final, re-suspender o worm sedimento em quantidade mínima (~ mL 500) de M9 e pipeta-los em placas NGM contendo um gramado de OP50 bactérias. Neste ponto, você não precisa mais trabalhar no bairro química. </li><li> Deixe o líquido secar por alguns minutos e em seguida, escolher e transferência de aparência saudável J4 larvas de placas semeadas frescas. Descarte a placa original de acordo com as diretrizes da instituição resíduos perigosos. </li><li> Deixe o auto worms mutagenizados fertilizar. Usando um microscópio estereoscópico, a tela do fenótipos geração F2 para mutantes recessivos de interesse. </li></ol> Parte 6: Psoralen mutagênese Nesta seção, vamos descrever o processo de mutagênese com TMP em conjunto com comprimento de onda ultravioleta longo (UV). TMP / UV método de mutagênese é amplamente utilizado para induzir mutações exclusão dentro de um genoma 3. <ol><li> Coletar worms 4-5, 6 cm de diâmetro placas de Petri em um tubo de centrifugação de 15 ml, conforme descrito no procedimento anterior. </li><li> Adicionar 10 ml M9, inverter 20 vezes a lavar os worms, e centrifugar a 1500 xg por 5 minutos. Desprezar o sobrenadante e repetir a lavagem para um total de 3 vezes. Aspirar o líquido e re-suspender a worms em cerca de 10 vezes o seu volume de M9 contendo 30 mg / ml de TMP. Pipeta mL 20 de TMP (3 mg / ml) em 2 ml de M9. Como EMS, TMP é um carcinógeno potente e deve ser tratada tomando medidas de segurança apropriadas. Para evitar de risco potencial do olho luz UV adequada usa deve ser usado. Os materiais descartáveis ​​devem ser descartados em sacos adequados biohazard. </li><li> Incubar o tubo no escuro por 15 minutos em temperatura ambiente em um rocker de baixa velocidade (40 UPM). Definir o tubo coberto verticalmente por 10 minutos para deixar tudo afundar o worms. </li><li> Remover os vermes do fundo do tubo com uma pipeta Pasteur e transferi-los para uma placa NGM unseeded. Desprezar o sobrenadante seguir as diretrizes da instituição químicos perigosos. </li><li> Manter a placa no escuro até o TMP excesso é absorvido pela placa. </li><li> Usando um medidor de intensidade de UV, definir a distância entre a fonte de UV de onda longa ea placa de tal forma que a intensidade na placa é de 500 μW / cm 2 4. Remova a tampa de alumínio ea tampa e cobrir e expor a placa contendo os vermes TMP tratados à radiação UV de onda longa por 50 segundos. </li><li> Cubra e deixe os vermes no escuro por 5 horas em temperatura ambiente. </li><li> Escolha e transferência de aparência saudável J4 larvas de placas semeadas frescas. Descarte a placa original após as diretrizes da instituição químicos perigosos. </li><li> Usando um microscópio estereoscópico, a tela do fenótipos geração F2 para a mutação de seu interesse. </li></ol> Parte 7: Resultados Representante Alguns dos P. fenótipos pacificus mutante resultante da mutagênese (seja por EMS ou TMP / UV) são mostrados na Figura 1. Cada mutante tem um fenótipo característico que é facilmente distinguível do animal do tipo selvagem. Do wormsque foram tratados com o mutagênico, 30 larvas J3/J4 da geração parental (P0) foram colocados em pratos separados e permitiu a pôr ovos, que são a geração F1. Das placas que mostrou progênie F1 saudável, um total de 1000 F1s foram transferidos para placas individuais ea progênie F2 foi exibido para os fenótipos. Animais com Dumpy 5,6, 6 e 7 descoordenada Transformer fenótipos foram encontrados. <img src="/files/ftp_upload/2293/2293fig1.jpg" alt="Figura 1" /> Figura 1. Pristionchus tipo pacificus selvagens e animais mutantes. A. wt wt hermafrodita masculino B. C. D. DPY mutante unc mutante E. tra mutante. Top dois painéis mostrando tipo selvagem hermafrodita (esquerda) e macho (à direita) animais para comparar com os mutantes diferentes nos painéis restantes.

<ol>
	<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=.">.</a> Genetics. 77 (1), 71-71 (1974).</li><li>Anderson, 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> Methods Cell Biol. 48, 31-31 (1995).</li><li>Yandell, M. D., Edgar, L. G., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (4), 1381-1381 (1994).</li><li>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=.">.</a> WormBook. , 1-1 (2005).</li><li>Kenning, C., Kipping, I., Sommer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Genesis. 40 (3), 176-176 (2004).</li><li>Sommer, R. J., Carta, L. K., Kim, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental and Applied Nematology. 19 (6), 511-511 (1996).</li><li>Pires-daSilva, A., 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=.">.</a> Genes &#38; development. 18 (10), 1198-1198 (2004).</li><li>Wei, A., Yuan, A., Fawcett, 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> Nucleic acids research. 30 (20), 110-110 (2002).</li><li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Rev Genet. 3 (5), 356-356 (2002).</li></ol>

Não há conflitos de interesse declarados.

Mutagênese é uma técnica amplamente empregada para estudos genéticos em vários organismos modelo experimental. Experimentos de mutagênese são freqüentemente usados ​​para identificar a função de um gene 2. Os procedimentos de mutagênese descritos aqui podem ser usados ​​não apenas para P. pacificus, mas também para C. elegans e de outras espécies de nematóides relacionados. Aqui nós descrevemos dois métodos, EMS e TMP / UV mutagênese. EM...

an introduction to worm lab: from culturing worms to mutagenesis

Este protocolo descreve os procedimentos para manter a nematóides em laboratório e como induz mutações los usando dois métodos alternativos: o metano sulfonato de etila (EMS) e 4, 5, 8-trimethylpsoralen combinada com luz ultravioleta (TMP / UV). Nematóides são poderosos sistemas biológicos para estudos genéticos por causa de seu plano corporal simples e sistema de acasalamento, que é composto de auto-fertilização hermafroditas e homens que podem gerar centenas de descendentes por animal. Nematóides são mantidos em placas de ágar contendo um gramado de bactérias e podem ser facilmente transferidos de uma placa para outra usando uma picareta. EMS é um agente alquilante comumente usado para induzir mutações pontuais e pequenas deleções, enquanto TMP / UV induz principalmente exclusões. Dependendo da espécie de nematóide sendo usado, as concentrações de EMS e TMP terá que ser otimizado. Para isolar mutações recessivas do nematóide pacificus Pristionchus, os animais da geração F2 foram selecionados para fenótipos visualmente. Para ilustrar esses métodos, mutagenizados worms e olhou para a falta de coordenação (Unc), Dumpy (DPY) e Transformer (Tra) mutantes.

Watch this Scientific Journal Video about An Introduction to Worm Lab: from Culturing Worms to Mutagenesis at JoVE.com

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Triagem de mutantes com defeitos fenotípicos é um método simples para identificar genes que funcionam em um determinado processo biológico. Neste artigo vamos descrever como worms cultura livre vivos (por exemplo,<em&gt; Pristionchus pacificus</em&gt;) No laboratório e mostrar dois métodos diferentes mutagênese, EMS e TMP UV /.

Uma Introdução ao Lab Worm: de Worms Cultivar a mutagênese

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

an-introduction-to-worm-lab-from-culturing-worms-to-mutagenesis

Research

JoVE Journal

Biology

34.2K visualizações.  University of Texas at Arlington. Triagem de mutantes com defeitos fenotípicos é um método simples para identificar genes que funcionam em um determinado processo biológico. Neste artigo vamos descrever como worms cultura livre vivos (por exemplo, Pristionchus pacificus) No laboratório e mostrar dois métodos diferentes mutagênese, EMS e TMP UV /.

Vídeo: An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Mutagênese do site caseiros Direção de Plasmids Whole

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Biologia

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Mutagênese Bissulfito Único de ovócitos

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

Introdução à membrana apoiada sólida baseada Eletrofisiologia

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

In vivo Reprogramação de células adultas somáticas a pluripotência por superexpressão de Fatores de Yamanaka

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Uma In vivo A reticulação Abordagem para isolar Complexos de proteínas a partir de Drosophila Os embriões

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

A cultura<em&gt; Caenorhabditis elegans</em &gt; Em Axenic Liquid Media e Criação de transgênicos Worms por bombardeamento com micropartículas

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Usando o rato do mamárias Células Tumorais para ensinar Biologia Conceitos Básicos: A Módulo Lab Simples

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Medindo a atividade enzimática de Lactase no laboratório de ensino

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Magnético-ativado da pilha classificação estratégias para isolar e purificar sinovial fluidos derivados mesenquimais células-tronco de um modelo de coelho

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolamento de miócitos murinos atrial e ventricular de alta qualidade para medidas simultâneas de Ca2+ transitórios e corrente de cálcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...