Este trabalho foi apoiado pelo Programa de Transferência adaptável e Seamless Tecnologia através de I &amp; D, o Japão Agência de Ciência e Tecnologia, Fundo de Promoção de pesquisa da ciência da Corporação de Promoção e Ajuda Mútua para Escolas Particulares do Japão Target-driven, e os números JSPS KAKENHI Grant 24300342 e 15H04313 .

1. Construção de um tipo de heptâmero sgRNA Biblioteca <ol><li> Selecionar um subconjunto dos 16.384 (4 7) sequências de 7 nt a menos que a biblioteca em grande escala deve ser construída. NOTA: As sequências podem ser aleatórios e / ou projetados para atingir RNAs celulares específicos. Para este último, encontrar estruturas em gancho de cabelo que se assemelha à do t-braço de ARNt num ARN alvo com o auxílio de um programa adequado de computador e / ou visualmente, e seleccione sequências complementares a sequências 7-nt imediatamente a jusante das estruturas em gancho de cabelo 4,10, 17,18. </li><li> Sintetizar cada um dos heptamers seleccionados como um 2&#39;-O totalmente -methylated, 5&#39;- e 3&#39;-ARN fosforilada com um sintetizador de ADN / ARN. Usar o método de fosforamidite sobre o vidro de poro controlado (CPG) de suporte, e deixar o 5&#39;-terminal de 4,4&#39;-dimetoxitritilo (DMT) no grupo protector no último ciclo (a adição de fosfato de 5 &#39;) da síntese 23. NOTA: sy personalizadonthetic ARN pode também ser obtido a partir de fornecedores de oligonucleótidos. </li><li> Após a conclusão do último ciclo de síntese, transferir a CPG com o oligonucleótido sintetizado a partir da coluna para um frasco de vidro com tampa de rosca-revestimento de Teflon, adiciona-se hidróxido de amónio a 1 ml de 29%, e incuba-se que a 55 ° C durante 8 h para clivar o oligonucleótido do CPG e remover os grupos protectores das bases e fosfatos. </li><li> Evapora-se hidróxido de amónio com um evaporador centrífugo a 40 ° C e re-dissolve-se o oligonucleótido em 0,2 ml de 0,1 M de n-hexilamónio de etilo. </li><li> Purifica-se o oligonucleótido sintético por meio de cromatografia líquida de alta eficiência de fase reversa utilizando uma coluna de poliestireno-divinilbenzeno com um tampão contendo 10-50% de acetonitrilo / M de acetato de n-hexilamónio 0,1. Executar a coluna a 2,0 ml / min durante 25 min a 80 ° C. </li><li> Adicionar 0,25-0,5 ml de ácido acético concentrado até a amostra fraccionado (1-2 mL) para fazer uma solução de ácido acético a 20%, e incubate esta solução durante 1 hora à temperatura ambiente para remover o grupo DMT. </li><li> Seca-se a amostra com um evaporador centrífugo a 40 ° C, re-dissolve-lo em hidróxido de amónio a 1 ml de 29%, e incubar a solução durante 15 minutos à temperatura ambiente para remover a cadeia lateral de fosfato 5 &#39;. </li><li> Reduzir o volume (1 ml) da amostra de cerca de 0,3 ml com um evaporador centrífugo a 40 ° C. </li><li> Dialisar toda a amostra concentrada contra água destilada (500 ml), utilizando um tubo de diálise com um peso molecular de corte de 1000 a 4 ° C durante a noite. </li><li> Transfira a amostra dialisada a partir do tubo de diálise para um tubo de 1,5 ml com uma pipeta, secá-lo com um evaporador centrífugo a 40 ° C, e, subsequentemente, re-dissolvê-lo em água-para-injecção de nível de água, o volume da qual deve ser pequena o suficiente para fazer uma solução maior do que 100 uM. </li><li> Medir uma densidade óptica da amostra de ARN a 260 nm com um espectrofotómetro e ajustar a sua concentração para 100uM por adição de água-para-injecção de nível de água. Guardar a solução de ARN a abaixo de -20 ° C antes da utilização. </li></ol> 2. Rastreio da Biblioteca sgRNA para a viabilidade celular <ol><li> Preparar 10 3 células / 99 ul / poço (por exemplo, HL60 e meio RPMI-8226) em um prato de 96 poços em meio RPMI-1640 suplementado com soro de bovino fetal a 10% e solução de penicilina-estreptomicina a 1%. Prepare poços contendo os meios de comunicação apenas para a subtracção de fundo. Para eliminar os efeitos de borda, adicione água estéril para esvaziar poços que cercam os poços de amostra. </li><li> Adicionar 1 ml de tipo heptâmero sgRNA (100 uM) dissolvido em água-para-injecção de nível de água a cada poço. Ensaiar cada sgRNA em triplicado. Incubar a placa a 37 ° C numa incubadora humidificada com 5% de CO 2 durante 3 dias. </li><li> Adicionar um volume apropriado de uma solução de WST-8 disponível comercialmente a cada poço. Incubar a placa a 37 ° C no mesmo incubador durante 1-4 horas. </li><li> Medir a absorvância at 450 nm com um leitor de microplacas. NOTA: Na maior parte dos casos, a linearidade entre a absorvância e o número de células viáveis ​​parece ser mantida a menos que o valor da absorvância superior a 1,0. </li></ol> 3. Avaliação da efectiva sgRNAs por citometria de fluxo <ol><li> Preparar 10 3 células / 99 ul / poço (por exemplo, HL60) em um prato de 96 poços em meio RPMI-1640 suplementado com soro de bovino fetal a 10% e solução de penicilina-estreptomicina a 1%. Preparar pelo menos 10 poços para uma sgRNA a ser testado. Para eliminar os efeitos de borda, adicione água estéril para esvaziar poços que cercam os poços de amostra. </li><li> Adicionar 1 ml de tipo heptâmero sgRNA (100 uM) dissolvido em água-para-injecção de nível de água a cada poço. Incubar a placa a 37 ° C numa incubadora humidificada com 5% de CO 2 durante 3 dias. </li><li> Recolher as células tratadas com o mesmo sgRNA de pelo menos 10 poços para um tubo de 5 ml com uma pipeta. Girar o tubo durante 5 min numa centrífuga a 300 xg, e descartar os meios de comunicação. </li><li> Adicionar 2 ml de 1x tampão fosfato salino (PBS) para o tubo, e re-suspender as células. Girar o tubo durante 5 min numa centrífuga a 300 xg, e descartar PBS. Adicionar 150 ul de 1x tampão de ligação da anexina V para o tubo, e re-suspender as células. </li><li> Adicionar 2 mL de ficoeritrina (PE) -conjugated solução anexina V e 1 ul de solução de 7-aminoactinomycin D (7AAD) para o tubo, mistura-los bem, e incuba-se o tubo durante 15 minutos à temperatura ambiente no escuro. Analisar a amostra usando um citômetro de fluxo 24. </li></ol>

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Niigata University of Pharmacy and Applied Life Sciences has a pending patent application on a subset of the heptamer-type sgRNAs.

In most cases, fully 2&#8242;-O-methylated, 5&#8242;- and 3&#8242;-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 &#181;M) appear to be stable at below -20 &#176;C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5&#8242;- or 3&#8242;-phosphate of a part of sgRNA molecules appears to be removed spontaneously du...

Silenciamento do gene VERDADEIRO (denominado após tR Nase Z L - L e tilizing fficacious silenciamento do gene) é uma das tecnologias de silenciamento do gene dirigida por RNA 1. Esta tecnologia foi desenvolvida com base nas propriedades de tRNase Z L (ou 3 &#39;tRNase), ARNt 3&#39; endorribonuclease processamento: ela pode clivar qualquer ARN alvo em qualquer sítio esperado sob a direcção de um pequeno ARN guias artificiais (sgRNA) através do reconhecimento de uma pré-ARNt ou de tipo micro-pré-ARNt-como a estrutura formada entre o RNA alvo e a sgRNA 2-9. sgRNA, que normalmente é 7-31 nt de comprimento, é categorizada em quatro grupos, 5&#39;-meia-ARNt, ARN heptâmero, ARN linear de 14 nt, e de ARN de gancho. Temos demonstrado a eficácia de silenciamento de genes VERDADEIRO através da introdução de vários sgRNAs artificialmente projetados em células vivas 10-15. Também mostrámos que sgRNAs pode ser absorvida pelas células Without quaisquer reagentes de transfecção e pode regular negativamente os seus níveis de RNA alvo e / ou induzir a apoptose em células cancerosas humanas 16 - 18. Silenciamento de genes VERDADEIRO aparece para trabalhar no intracelular e sistema de rede de transcrição intercelular via tRNase Z L e sgRNAs naturais 19 - 21. Construímos uma biblioteca sgRNA contendo 156 do tipo heptâmero sgRNAs para procurar potenciais sgRNAs do tipo heptâmero terapêuticas para cancros do sangue 22. Temos rastreados para que o efeito sobre a viabilidade de células de mieloma e de leucemia humana, e descobriram que 20 deles pode eficientemente induzir a apoptose em pelo menos uma das linhas celulares de cancro. E quatro deles foram mostrados para reduzir as taxas de crescimento de tumor em experiências de xenoenxerto de ratinho. Aqui apresentamos um protocolo para o rastreio de uma biblioteca sgRNA tipo heptâmero para os potenciais drogas terapêuticas contra o câncer no sangue. O protocolo inclui comopara construir a biblioteca sgRNA, a forma de avaliar o efeito de cada sgRNA sobre a viabilidade celular, e a forma de avaliar ainda mais os sgRNAs eficazes por citometria de fluxo. Análise para ARN alvo celular da sgRNA e avaliação de sgRNAs eficazes em modelos de xenoenxerto de ratinho pode ser realizada de acordo com métodos convencionais 17,22, embora estes não estão incluídas no presente protocolo.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Custom heptamer RNA</td>
			<td style="width:167px;">Nippon Bioservice</td>
			<td style="width:144px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OligoSep Prep HC Cartridge</td>
			<td style="width:167px;">Transgenomic</td>
			<td style="width:144px;">99-3860</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water-for-injection-grade Water</td>
			<td style="width:167px;">Otsuka Pharmaceutical&#160;</td>
			<td style="width:144px;">7131400A2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 medium</td>
			<td style="width:167px;">Wako</td>
			<td style="width:144px;">189-02025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">172012-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Penicillin-Streptomycin Mixed Solution</td>
			<td style="width:167px;">Nacalai Tesque</td>
			<td style="width:144px;">26253-84</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well Plate</td>
			<td style="width:167px;">Greiner Bio-one</td>
			<td style="width:144px;">655 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Counting Kit-8</td>
			<td style="width:167px;">DOJINDO</td>
			<td style="width:144px;">343-07623</td>
			<td>WST-8 solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate Reader, Sunrise Thermo RC-R</td>
			<td style="width:167px;">TEKAN</td>
			<td style="width:144px;">510-82851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Round-Bottom Tube</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:144px;">60819-820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">P7059-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V Binding Buffer</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-66121E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-65875X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7AAD</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">A9400-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer, FACSCalibur</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">342973</td>
			<td></td>
		</tr>
	</tbody>
</table>

true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents

Silenciamento do gene VERDADEIRO (denominado após tR Nase Z L - L tilizing e fficacious silenciamento do gene) é um do gene dirigida por ARN de silenciamento tecnologias, que utiliza uma pequena guia de ARN artificial (sgRNA) para guiar endorribonuclease processamento ARNt 3 &#39;, tRNase Z L , para reconhecer um alvo de ARN. sgRNAs podem ser absorvidos pelas células sem quaisquer reagentes de transfecção e pode regular negativamente os seus níveis de RNA alvo e / ou induzir a apoptose em células cancerosas humanas. Temos rastreada uma biblioteca sgRNA contendo 156 sgRNAs do tipo heptâmero para o efeito sobre a viabilidade de células de mieloma e de leucemia humana, e descobriram que 20 deles pode eficientemente induzir a apoptose em pelo menos uma das linhas celulares de cancro. Aqui apresentamos um protocolo para o rastreio de uma biblioteca sgRNA tipo heptâmero para os potenciais drogas terapêuticas contra o câncer no sangue. O protocolo inclui a forma de construir a biblioteca sgRNA, a forma de avaliar o efeito de cada sgRNA sobre a viabilidade celular, e a forma de further avaliar os sgRNAs eficazes por citometria de fluxo. Cerca de 2000 acessos seria esperado para ser obtido por rastreio da biblioteca em grande escala sgRNA composto de 16.384 heptamers.

Os seis tipo heptâmero sgRNAs 5&#39;-AUCUUCA-3 &#39;(H1885), 5&#39;-ACACACA-3&#39; (H3277), 5&#39;-GGGGGCG-3 &#39;(H10927), 5&#39;-GGGGCCC-3&#39; (H10944), 5&#39;-GCCCCCG-3 &#39;(H12287), e 5&#39;-CACCAGC-3&#39; (H13260) foram sintetizados quimicamente como 2&#39;-O-metil ARN contendo 5&#39;- e 3&#39;-fosfatos. Estes sgRNAs foram examinados quanto aos efeitos sobre a viabilidade de uma linha celular de leucemia human...

Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Aqui apresentamos um protocolo para o rastreio de uma biblioteca sgRNA tipo heptâmero para os potenciais drogas terapêuticas contra o câncer no sangue.

VERDADEIRO silenciamento gênico: Triagem de um heptâmero tipo Guia de pequeno RNA Biblioteca de agentes terapêuticos potenciais Câncer

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

true-gene-silencing-screening-heptamer-type-small-guide-rna-library

Research

JoVE Journal

Medicine

5.6K visualizações.  Niigata University of Pharmacy and Applied Life Sciences.  Aqui apresentamos um protocolo para o rastreio de uma biblioteca sgRNA tipo heptâmero para os potenciais drogas terapêuticas contra o câncer no sangue. 

Vídeo: TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Termoplastia brônquica: uma nova abordagem terapêutica para Asma Grave

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Medicina

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Ortotópico do xeno-Human-luciferase Tagged maligna dos nervos periféricos Células Tumorais Bainha para In vivo Teste de Agentes Terapêuticos candidatos

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

Entrega de agentes terapêuticos Através intracerebroventricular (ICV) e injecção intravenosa (IV) em ratos

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

Um método simples parafuso Guia para intracraniana Estudos Xenoenxerto em Ratos

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

DNA Vector baseado em RNA de interferência para estudar a função Gênica em Câncer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Uma nova abordagem cirúrgica para administração intratraqueal de agentes bioativos em um modelo de camundongo Fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Triagem para Modificadores Melanoma usando um modelo de tumor Zebrafish autóctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Um modelo de cancro da bexiga para Ortotópico Gene Estudos de entrega

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

A plataforma de triagem baseada luciferase diversificadas para interrogar associados ao câncer transdução de sinal em células cultivadas

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Concentre Formação: Um Ensaio Celular para Determinar o Potencial oncogénica de um Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...