Somos gratos aos Serviços Ambientais Association Education Trust, a Biologia Sintética em Cambridge fundo Synbio e do Ministério da Justiça Social e Empoderamento, Governo da Índia, para apoio financeiro.

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antialgal,+antibacterial+and+antifungal+activity+of+two+metabolites+produced+and+excreted+by+cyanobacteria+during+growth.">Antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth.</a> Microbiol Res. 161, 180-186 (2006).</li><li>Scott, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiesel+from+algae:+challenges+and+prospects.">Biodiesel from algae: challenges and prospects.</a> Curr Opin Biotechnol. 21, 277-286 (2010).</li><li>Lea-Smith, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycobilisome-deficient+strains+of+Synechocystis+sp.+PCC+6803+have+reduced+size+and+require+carbon-limiting+conditions+to+exhibit+enhanced+productivity.">Phycobilisome-deficient strains of Synechocystis sp. PCC 6803 have reduced size and require carbon-limiting conditions to exhibit enhanced productivity.</a> Plant Physiol. 165, 705-714 (2014).</li><li>Lea-Smith, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thylakoid+terminal+oxidases+are+essential+for+the+cyanobacterium+Synechocystis+sp.+PCC+6803+to+survive+rapidly+changing+light+intensities.">Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities.</a> Plant Physiol. 162, 484-495 (2013).</li><li>Liu, X., Sheng, J., Curtiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+production+in+genetically+modified+cyanobacteria.">Fatty acid production in genetically modified cyanobacteria.</a> Proc Natl Acad Sci U S A. 108, 6899-6904 (2011).</li><li>Xu, H., Vavilin, D., Funk, C., Vermaas, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+deletions+of+small+cab-like+proteins+in+the+cyanobacterium+Synechocystis+sp+PCC+6803+-+Consequences+for+pigment+biosynthesis+and+accumulation.">Multiple deletions of small cab-like proteins in the cyanobacterium Synechocystis sp PCC 6803 - Consequences for pigment biosynthesis and accumulation.</a> J Biol Chem. 279, 27971-27979 (2004).</li><li>Castenholz, R. 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The authors declare that they have no competing financial interests.

Os passos mais críticos na geração de mutantes não marcados são: 1) um projeto cuidadoso plasmídeo para garantir que apenas a região de destino é alterada; 2) garantir que as amostras permanecem axênico, especialmente quando cultivadas em sacarose; 3) para a geração de células chapeamento mutante marcada inicialmente transformado em placas de agar BG11 falta antibióticos, seguido por adição de ágar antibióticos mais 24 horas mais tarde; 4) cultura marcada mutantes para 4 dias completos antes de plaqueam...

As cianobactérias são um filo antiga e diversificada evolutivamente de bactérias encontradas em quase todos ambiente natural na Terra. Em ecossistemas marinhos são particularmente abundantes e desempenham um papel fundamental em muitos ciclos de nutrientes, sendo responsável por cerca de metade da fixação de carbono 1, a maioria da fixação de nitrogênio 2 e centenas de milhões de toneladas de produção de hidrocarbonetos 3 nos oceanos anualmente. Cloroplastos, a organela responsável pela fotossíntese em algas e plantas eucariótica, são susceptíveis de ter evoluído de uma cianobactéria que foi engolida por um organismo hospedeiro 4. As cianobactérias provaram organismos modelo útil para o estudo da fotossíntese, transporte de electrões 5 e vias bioquímicas, muitos dos quais são conservados em plantas. Em adição cianobactérias são cada vez mais utilizados para a produção de alimentos, biocombustíveis 6, 7 e electricidade industriais compostos 8, devido à sua oiconversão ghly eficiente de água e CO 2 a biomassa usando a energia solar 9. Muitas espécies podem ser cultivadas em terras não-aráveis ​​com nutrientes mínimos e água do mar, o que sugere que as cianobactérias potencialmente poderiam ser cultivadas em grande escala, sem afetar a produção agrícola. Certas espécies também são fontes de produtos naturais, incluindo antifúngicos, antibacterianos e anti-câncer compostos 10,11. A capacidade de gerar mutantes é a chave para a compreensão de cianobactérias fotossíntese, bioquímica e fisiologia, e essencial para o desenvolvimento de cepas para fins industriais. A maioria dos trabalhos publicados produzir estirpes geneticamente modificados por inserção de uma cassete de resistência a antibiótico no local de interesse. Isto limita o número de mutações que podem ser introduzidas numa estirpe, como apenas algumas cassetes de resistência aos antibióticos estão disponíveis para uso em cianobactérias. Estirpes que contêm genes que conferem re antibióticosistance não pode ser usado para a produção industrial em lagoas abertas, o que é susceptível de ser o único meio de baixo custo para a produção de biocombustíveis e outros produtos de baixo valor 12. A geração de mutantes sem marcação supera essas limitações. mutantes não marcadas não contêm DNA estranho, a não ser intencionalmente incluídos, e pode ser manipulado diversas vezes. Por isso, é possível gerar o maior número de alterações em uma cepa como desejado. Além disso, os efeitos polares de genes a jusante do local de modificação pode ser minimizado, permitindo a modificação mais preciso do organismo 13. Para gerar estirpes mutantes, plasmídeos suicidas contendo dois fragmentos de ADN idênticos às regiões no cromossoma de cianobactérias que flanqueiam o gene a ser suprimido (denominado a 5 &#39;e 3&#39; que flanqueiam regiões) são construídos em primeiro lugar. Dois genes são então inserida entre estas regiões flanqueadoras. Uma delas codifica uma proteína de resistência a antibiótico; o segundo codifica SacB, que produces levanossacarase, um composto que confere sensibilidade à sacarose. Na primeira fase do processo, os mutantes marcadas, as estirpes ou seja, contendo algum ADN estranho, são gerados. A construção de plasmídeo é misturado com as células de cianobactérias e o ADN é tomado naturalmente pelo organismo. Os transformantes são seleccionados por crescimento em placas de agar contendo os antibióticos apropriados e o genótipo mutante verificada por PCR. plasmídeos suicidas não pode replicar dentro da estirpe de interesse. Portanto, quaisquer colónias resistentes ao antibiótico irá resultar a partir de um evento de recombinação em que o gene de interesse é inserida no cromossoma. Para gerar mutantes não marcados, o mutante marcada é, em seguida, misturado com um segundo plasmídeo suicida contendo apenas as regiões f lanqueadoras 5 &#39;e 3&#39;. No entanto, se for necessária a inserção de ADN estranho, num plasmídeo que consiste em 5 &#39;e 3&#39; com regiões de uma cassete que contém os genes de interesse inserido entre estes fragmentos de ADN de flanqueamento, pode ser utilizado. Selecção é através de crescimento em placas de agar contendo sucrose. Como a sacarose é letal para as células quando o produto do gene sacB é expresso, as únicas células que sobrevivem são aquelas nas quais ocorreu um segundo evento de recombinação, em que o gene de sensibilidade de sacarose, em adição ao gene de resistência aos antibióticos, foi recombinado para fora do cromossoma e para o plasmídeo. Como consequência da permuta de recombinação, as regiões f lanqueadoras e qualquer ADN entre eles são inseridos no cromossoma. Temos utilizado com sucesso destes métodos para gerar múltiplas mutações cromossómicas na mesma estirpe de Synechocystis sp. PCC6803 (doravante referida como Synechocystis) 13,14, para introduzir mutações pontuais em um gene de interesse 13 e para a expressão de cassetes de genes. Enquanto a geração de nocautes não marcado foi demonstrada antes de nosso trabalho em Synechocystis 15,16, um método detalhado, auxiliado poruma apresentação visual dos passos críticos, não está disponível publicamente. Nós também aplicaram o mesmo método para geração de nocautes marcados em outro cianobactéria modelo, Synechococcus sp. PCC7002 (doravante referida como Synechococcus). Este protocolo fornece um método claro, simples para a geração de mutantes e um protocolo rápido para validar e armazenar estas estirpes.

<table border="0" cellpadding="0" cellspacing="0" width="645">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">NaNO3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">S5506</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4.7H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">230391</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">citric acid</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">C0759</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2EDTA</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">EDT002</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">H3BO3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">339067</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MnCl2.4H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">M3634</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ZnSO4.7H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">Z4750</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2MoO4.2H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">331058</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CuSO4.5H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">209198</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Co(NO3)2.6H2O</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">239267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ferric ammonium citrate</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">F5879</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">K2HPO4</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">P3786</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SODC001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">T1375</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SODH001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cyanocobalamin</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">47869</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2S2O3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">72049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Bacto agar</td>
			<td style="width:123px;">BD</td>
			<td style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Sucrose</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">SUC001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Petri dish 90 mm triple vented</td>
			<td style="width:123px;">Greiner</td>
			<td style="width:119px;">633185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 &#181;m filters</td>
			<td style="width:123px;">Sartorius</td>
			<td style="width:119px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">100 mL conical flasks</td>
			<td style="width:123px;">Pyrex</td>
			<td style="width:119px;">CON004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Parafilm M 100 mm x38 m</td>
			<td style="width:123px;">Bemis</td>
			<td style="width:119px;">FIL003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phusion high fidelity DNA polymerase&#160;</td>
			<td style="width:123px;">Phusion</td>
			<td style="width:119px;">F-530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td style="width:123px;">Melford</td>
			<td style="width:119px;">MB1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA purification kit&#160;</td>
			<td style="width:123px;">MoBio</td>
			<td style="width:119px;">12100-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Restriction endonucleases</td>
			<td style="width:123px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">T4 ligase</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">EL0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Luria Bertani broth</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">12795-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">MES</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">M8250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Kanamycin sulfate</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">60615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ampicillin</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">GeneJET plasmid miniprep kit</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:119px;">K0503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 mL round-bottom tube</td>
			<td style="width:123px;">BD falcon</td>
			<td style="width:119px;">352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">GoTaq G2 Flexi DNA polymerase</td>
			<td style="width:123px;">Promega</td>
			<td style="width:119px;">M7805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">425-600 &#181;m glass beads</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glycerol</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DMSO</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fluorescent bulbs</td>
			<td style="width:123px;">Gro-Lux</td>
			<td style="width:119px;">69</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">HT multitron photobioreactor</td>
			<td style="width:123px;">Infors</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of marked and markerless mutants in model cyanobacterial species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

O plasmídeo concepção é crítica para a geração bem sucedida de ambos os mutantes marcadas e não marcadas. A Figura 1 dá um exemplo do plasmídeo A e B usadas para gerar um mutante de deleção nos genes de Synechocystis cpcC1 e cpcC2 13. Em cada caso, as regiões f lanqueadoras 5 &#39;e 3&#39; são aproximadamente 900-1.000 pb. flanqueando reduzidas pode ser utilizado, embora o menor temos testadas com sucesso tem sido aprox...

Watch this Scientific Journal Video about Generation of Marked and Markerless Mutants in Model Cyanobacterial Species at JoVE.com

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Geração de mutantes sem marcadores marcados e no modelo de cianobactérias da espécie

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

generation-marked-markerless-mutants-model-cyanobacterial

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.9K visualizações.  University of Cambridge. Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Vídeo: Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Ensaios Neurocircuit para Apreensões em mutantes de Epilepsia Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Biologia

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Transplante de lente em Zebrafish e sua Aplicação na Análise de Mutantes Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

A técnica de amostragem não invasiva cabelo obter DNA de alta qualidade a partir de Elusive Pequenos Mamíferos

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Geração, purificação, caracterização e de Cell-invasivos espécies de proteína Disc1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Detectando, visualizar e quantificar a geração de espécies reativas de oxigênio em um modelo de sistema Amoeba

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Rápida e eficiente Zebrafish Genotipagem Usando PCR com alta resolução Análise Melt

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Eficiente Geração induzida pelo homem células-tronco pluripotentes a partir de células somáticas humanas com Sendai-vírus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Clonal Rastreamento de Stem hematopoiéticas e células progenitoras Marcado por cinco proteínas fluorescentes usando confocal e microscopia multifotônica

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

A Assembleia e Aplicação de &#39;tesoura Anéis&#39;: A endotelial novo modelo de Orbital, unidirecional e periódica fluxo de fluido e tensão de cisalhamento

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Análise de microbiota usando PCR de duas etapas e sequenciamento do gene 16S rRNA de próxima geração

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Geração de mutantes sem marcadores marcados e no modelo de cianobactérias da espécie

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