Damos las gracias a James EK Hildreth, Meharry Medical College, por su asesoramiento y revisión de este manuscrito. También queremos agradecer a Diana Marver para la edición del manuscrito. Este trabajo fue apoyado por el Centro de Meharry Vanderbilt para la Investigación del SIDA (NIH P30 AI054999-05), el Centro de Salud SIDA disparidades de Investigación, NIH RR019192 5U54-05, el Centro de Meharry de Investigación Clínica, de Subvenciones del NIH P20RR011792, y por el NIH conceder P01 CA113239. DJA fue parcialmente financiado por las subvenciones piloto del Centro Vanderbilt-Meharry for AIDS Research (CFAR) y el Centro para la Investigación Clínica Meharry (CRC).

<ol>
	<li>Sitas, F., Newton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma+in+South+Africa.">Kaposi's sarcoma in South Africa.</a> J Natl Cancer Inst Monogr. 28, 1-4 (2001).</li><li>Moore, P. S., Chang, Y., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma+associated+herpesvirus.">Kaposi's sarcoma associated herpesvirus.</a> Field's Virology. , 2803-2833 (2002).</li><li>Chang, Y., Cesarman, E., Pessin, M. S., Lee, F., Culpepper, J., Knowles, D. M., Moore, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+herpesvirus-like+DNA+sequences+in+AIDS-associated+Kaposi's+sarcoma.">Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.</a> Science. 266, 1865-1869 (1994).</li><li>Cesarman, E., Chang, Y., Moore, P., Said, J. W., Knowles, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma-associated+herpesvirus-like+DNA+sequences+in+AIDS-related+body-cavity-based+lymphomas.">Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas.</a> N Engl J Med. 18, 1186-1191 (1995).</li><li>Cesarman, E., Knowles, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma-associated+herpesvirus:+a+lymphotropic+human+herpesvirus+associated+with+Kaposi's+sarcoma,+primary+effusion+lymphoma,+and+multicentric+Castleman's+disease.">Kaposi's sarcoma-associated herpesvirus: a lymphotropic human herpesvirus associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease.</a> Semin Diagn Pathol. 14, 54-66 (1997).</li><li>Chiou, C. J., Poole, L. J., Kim, P. S., Ciufo, D. M., Cannon, J. S., ap Rhys, C. M., Alcendor, D. J., Zong, J. C., Ambinder, R. F., Hayward, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+gene+expression+and+a+transactivation+function+exhibited+by+the+vGCR+(ORF74)+chemokine+receptor+protein+of+Kaposi's+sarcoma-associated+herpesvirus.">Patterns of gene expression and a transactivation function exhibited by the vGCR (ORF74) chemokine receptor protein of Kaposi's sarcoma-associated herpesvirus.</a> J Virol. 76, 3421-3439 (2002).</li><li>Staskus, K. A., Zhong, W., Gebhard, K., Herndier, B., Wang, H., Renne, R., Beneke, J., Pudney, J., Anderson, D. J., Ganem, D., Haase, A. T., T, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma-associated+herpesvirus+gene+expression+in+endothelial+(spindle)+tumor+cells.">Kaposi's sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells.</a> J Virol. 71, 715-719 (1997).</li><li>Alcendor, D. J., Zhu, W. Q., Desai, P., Vigil, H. E., Hayward, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+downregulation+of+galectin-3+in+kaposi's+sarcoma.">KSHV downregulation of galectin-3 in kaposi's sarcoma.</a> Glycobiology. , (2009).</li><li>Ciufo, D. M., Cannon, J. S., Poole, L. J., Wu, F. Y., Murray, P., Ambinder, R. F., Hayward, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle+cell+conversion+by+Kaposi's+sarcoma-associated+herpesvirus:+formation+of+colonies+and+plaques+with+mixed+lytic+and+latent+gene+expression+in+infected+primary+dermal+microvascular+endothelial+cell+cultures.">Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures.</a> J Virol. 75, 5614-5626 (2001).</li><li>Long, E., Ilie, M., Hofman, V., Havet, K., Selva, E., Butori, C., Lacour, J. P., Nelson, A. M., Cathomas, G., Hofman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LANA-1,+Bcl-2,+Mcl-1+and+HIF-1alpha+protein+expression+in+HIV-associated+Kaposi+sarcoma.">LANA-1, Bcl-2, Mcl-1 and HIF-1alpha protein expression in HIV-associated Kaposi sarcoma.</a> Virchows Arch. 2, 159-170 (2009).</li></ol>

Las metodologías utilizadas en este informe se puede realizar en una variedad de entornos de laboratorio. Que requieren algún equipo especial o acceso a instalaciones básicas. Las debidas precauciones se deben tomar al hacer experimentos con virus patógenos infecciosos como HVSK que puede requerir el uso de un nivel biosaftey 3 (BSL-3) instalaciones. Adquisiciones de los tejidos también debe ser aprobado por las juntas de revisión institucional adecuado (IRB). Estas metodologías en co...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>05-0001DJ</td>
<td>&nbsp;</td>
<tr>
<td>TPA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1585</td>
<td>&nbsp;</td>
<tr>
<td>PBS pH 7.4</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10010-023</td>
<td>&nbsp;</td>
<tr>
<td>Sodium butyrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>19364 (FLUKA)</td>
<td>&nbsp;</td>
<tr>
<td>EBM-2 media</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-3156</td>
<td>+ bullet kits supplement </td>
</tr>
<tr>
<td>DMVEC cells (HMVEC)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC2543</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30066.03</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>154461</td>
<td>&nbsp;</td>
<tr>
<td>KSHV LANA Mab</td>
<td>&nbsp;</td>
<td>Vector Labs</td>
<td>VP-H913</td>
<td>1:50 dilution/IFA</td>
</tr>
<tr>
<td>Galectin-3 goat pAb</td>
<td>&nbsp;</td>
<td>R&D Systems</td>
<td>AF1154</td>
<td>&nbsp;</td>
<tr>
<td>Donkey-anti-goat FITC</td>
<td>&nbsp;</td>
<td>Jackson ImmunoRessearch</td>
<td>705-095-1</td>
<td>&nbsp;</td>
<tr>
<td>Donkey-anti-goat Rho</td>
<td>&nbsp;</td>
<td>Jackson ImmunoRessearch</td>
<td>705-295-003</td>
<td>1:100 dilution/IFA</td>
</tr>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>Vector Labs</td>
<td>H 1500</td>
<td>Vectashield with DAPI</td>
</tr>
<tr>
<td>BCA Protein Assay Kit</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>23235</td>
<td>&nbsp;</td>
<tr>
<td>Nitrocellulose </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0112</td>
<td>&nbsp;</td>
<tr>
<td>Donkey-anti-goat-conj. </td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>SC 2020</td>
<td>&nbsp;</td>
<tr>
<td>Western substrate</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>34075</td>
<td>&nbsp;</td>
<tr>
<td>Biotin-rabbit-anti goat</td>
<td>&nbsp;</td>
<td>DAKO</td>
<td>305-065-045</td>
<td>&nbsp;</td>
<tr>
<td>AP-Strepavidin conj.</td>
<td>&nbsp;</td>
<td>DAKO</td>
<td>K 0492 (kit)</td>
<td>&nbsp;</td>
<tr>
<td>RNase DNase Free Set</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>79254</td>
<td>&nbsp;</td>
<tr>
<td>MJ Mini Cycler</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>PTC-1148C</td>
<td>&nbsp;</td>
<tr>
<td>Bio-Photometer</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>952000006</td>
<td>&nbsp;</td>
<tr>
<td>RC 6 Plus Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>46910</td>
<td>&nbsp;</td>
<tr>
<td>Coulter Optima L-90K Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>392052</td>
<td>&nbsp;</td>
<tr>
<td>MYiQ iCycler Real Time PCR Detection System</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-9770</td>
<td>&nbsp;</td>
<tr>
<td>TE 2000S Fluorescent microscope</td>
<td>&nbsp;</td>
<td>NIKON</td>
<td>TE2000S</td>
<td>&nbsp;</td>
<tr>
<td>2100 Bioanalyzer</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2938C</td>
<td>&nbsp;</td>
<tr>
<td>Eclipse E 200</td>
<td>&nbsp;</td>
<td>NIKON</td>
<td>E 200</td>
<td>&nbsp;</td>
<tr>
<td>Sorvall Legend RT Centrifuge</td>
<td>&nbsp;</td>
<td>Thermo</td>
<td>75004377</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

identifying dysregulated genes induced by kaposi's sarcoma-associated herpesvirus (kshv)

Actualmente KS es el más predominante del VIH / SIDA relacionados con tumores malignos en el sur de África y de ahí el mundo. 1,2 Se caracteriza por ser un tumor angioproliferative de las células endoteliales vasculares y produce enfermedades raras células B linfoproliferativos en forma de linfomas derrame pleural (PEL) y algunas formas de enfermedad de Castleman multicéntrica. 5.3 Sólo 1.5% de las células KS lesiones apoyar activamente la replicación lítica del sarcoma de Kaposi asociado al virus del herpes (HVSK), el agente etiológico asociado con sarcoma de Kaposi, y está claro que los factores celulares que interactuar con factores virales en el proceso de oncogénesis y la progresión del tumor 6,7. Identificación de la novela factor del huésped determinantes que contribuyen a la patología de KS es esencial para el desarrollo de marcadores de pronóstico para la progresión tumoral y la metástasis, así como para el desarrollo de nuevas terapias para el tratamiento del sarcoma de Kaposi 8. Los detalles del vídeo que acompaña a los métodos que utilizamos para identificar a la célula huésped programas de expresión de genes alterados en las células endoteliales microvasculares dérmicas (DMVEC) después de la infección HVSK y en el tejido tumoral KS. 9 Una vez que los genes desregulados son identificados por análisis de microarrays, los cambios en la expresión de la proteína se confirmada por inmunoblot y la inmunofluorescencia doble etiquetado. Cambios en la expresión transcripcional de genes desregulados se confirmó in vitro por cuantitativa en tiempo real de reacción en cadena de la polimerasa (QRT-PCR). La validación de los resultados in vitro usando tejidos de archivo tumor KS también se realiza por inmunohistoquímica doble etiquetado y de microarrays de tejidos 8,10. Nuestro enfoque para identificar los genes que están alteradas en el microambiente del tejido del tumor KS permitirá el desarrollo de la in vitro y, posteriormente, en los sistemas modelo in vivo para descubrimiento y evaluación de nuevos terapéuticos potenciales para el tratamiento del sarcoma de Kaposi.

Watch this Scientific Journal Video about Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV at JoVE.com

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Factores de la célula huésped juegan un papel crítico en el establecimiento y mantenimiento de sarcoma de Kaposi (SK). Resumimos los métodos para identificar los factores de la célula huésped alterada en las células infectadas por HVSK DMVEC, y en el tejido tumoral KS. Genes celulares alteradas por el virus servirá como posible objetivo (s) de nuevas terapias.

La identificación de genes inducida por la desregulación de Sarcoma de Kaposi asociado al virus del herpes (HVSK)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

identifying-dysregulated-genes-induced-kaposi-s-sarcoma-associated

Research

JoVE Journal

Immunology and Infection

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

12.5K vistas.  Meharry Medical College. Factores de la célula huésped juegan un papel crítico en el establecimiento y mantenimiento de sarcoma de Kaposi (SK). Resumimos los métodos para identificar los factores de la célula huésped alterada en las células infectadas por HVSK DMVEC, y en el tejido tumoral KS. Genes celulares alteradas por el virus servirá como posible objetivo (s) de nuevas terapias.

Video: Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

Transfección y mutagénesis de los genes diana en células de mosquito por nucleico cerrado modificado ácido oligonucleótidos

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

Investigación

Inmunología e Infección

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Disección de Host-virus Interacción en la replicación lítica de un Herpesvirus Modelo

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

La evaluación de la hipermutación somática en Ramos células B después de sobreexpresión o derribo de genes específicos

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

La identificación de mutaciones del ADN en Purificada hematopoyéticas células madre / progenitoras

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

El uso de ARN de interferencia para Investigar la respuesta inmune innata en macrófagos de ratón

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Análisis rápido y robusto de Celular y Molecular Polarización Inducida por Chemokine Señalización

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

Desarrollo y validación de un método de PCR cuantitativa para Equid herpesvirus-2 Diagnóstico en los flujos respiratorios

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

La cuantificación rápida de Blastogénesis inducida por mitógeno de linfocitos T para identificar fármacos inmunomoduladores

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Efectos de la exposición de formaldehído a un modelo de rata de la Dermatitis atópica inducida por el tratamiento Neonatal de capsaicina

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un método para evaluar las funciones efectoras mediadas por Fc inducidas por los anticuerpos específicos de la hemaglutinina de la gripe

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...