Idea?

Este estudio utiliza Chip para mapear sitios y genes ubiquitinados en el genoma
http://www.signaling-gateway.org/update/updates/200805/nmeth0508-380.html
Podria hacerse algo parecido con los factores de splicing? Quiza para mapear regulacion trans de splicing despues de estimulos especificos?

Nuevo mecanismo? Activacion por 5 UTRs.

PLOS Medicine

A Novel Tumor-Promoting Function Residing in the 5′ Non-coding Region of vascular endothelial growth factor mRNA

Kiyoshi Masuda, Shigetada Teshima-Kondo^*, Mina Mukaijo, Naoko Yamagishi, Yoshiko Nishikawa, Kensei Nishida, Tomoko Kawai, Kazuhito Rokutan

Background

Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells.

Methods and Findings

Knockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF₁₆₅ did not completely inhibit this apoptosis. Conversely, overexpression of VEGF₁₆₅ increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF₁₆₅-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5′UTRs. Using these plasmids, we revealed that the 5′UTR of vegf mRNA possessed anti-apoptotic activity. The 5′UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5′UTR or the mutated 5′UTR. The clones expressing the 5′UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5′UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5′UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)α therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNα responses.

Conclusions

These findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.

Valdría la pena probar?

Pimp Your Plasmid Growth Medium

April 28, 2008 | By Nick In Tech Tips |

I often wonder why it is that molecular biology researchers stubbornly refuse to change 40 year old methods that, while they work, are not as good as newer, faster and cheaper methods out there.

I suppose rational scientists have often irrational superstitions.

One example of an old method that could be improved is the growth media used for plasmid preparation.

The majority of us, throughout our university careers, have used either SOC, LB or TB, for recombinant plasmid propagation, typically in E. coli. LB or Luria-Bertani broth has been in use for almost 60 years or thereabouts, while SOC has certainly been in use for 2 decades.

But by adding in a few more ingredients or being more economical on others (especially yeast extract and tryptone) that you could get a higher plasmid yield, quicker and with less money.

To counter the naysayers, nobody wants to make very complex with 15 ingredients requiring filter sterilisation, as this obviously defeats the object of economy of time and budget. Indeed, there are trade-offs between optimising for biomass, plasmid yield, quality, stability and cost with the difference between protein production and plasmid production being that plasmid production requires only cell growth, division, and plasmid stability.

The good news is that Michael Danquah and Gareth Forde from Monash University down-under have devised a stoichiometrically optimised medium for plasmid production. PDM, supposedly yields under the conditions they tested, twice the amount of plasmid in both volumetric and specific yields compared to TB , LB is left in the dust. Better yet, because it uses less tryptone and yeast extract, the cost per mg of DNA is roughly one quarter compared to LB.

The recipes for LB, TB, SOC and PDM are shown below. If you decide to break with tradition and give PDM a go, be sure to tell us how it goes.

Note – Autoclave glucose, KH₂PO₄ and Na₂HPO₄ separately

Peligros de la contaminacion con micoplasma

Oncogene advance online publication 14 April 2008; doi: 10.1038/onc.2008.103

Mycoplasma infection suppresses p53, activates NF-B and cooperates with oncogenic Ras in rodent fibroblast transformation

Prokaryotes of the genus Mycoplasma are the smallest cellular organisms that persist as obligate extracellular parasites. Although mycoplasma infection is known to be associated with chromosomal instability and can promote malignant transformation, the mechanisms underlying these phenomena remain unknown. Since persistence of many cellular parasites requires suppression of apoptosis in host cells, we tested the effect of mycoplasma infection on the activity of the p53 and nuclear factor (NF)-B pathways, major mechanisms controlling programmed cell death. To monitor the activity of p53 and NF-B in mycoplasma-infected cells, we used a panel of reporter cell lines expressing the bacterial -galactosidase gene under the control of p53- or NF-B-responsive promoters. Cells incubated with media conditioned with different species of mycoplasma showed constitutive activation of NF-B and reduced activation of p53, common characteristics of the majority of human tumor cells, with M. arginini having the strongest effect among the species tested. Moreover, mycoplasma infection reduced the expression level and inducibility of an endogenous p53-responsive gene, p21^waf1, and inhibited apoptosis induced by genotoxic stress. Infection with M. arginini made rat and mouse embryo fibroblasts susceptible to transformation with oncogenic H-Ras, whereas mycoplasma-free cells underwent irreversible p53-dependent growth arrest. Mycoplasma infection was as effective as shRNA-mediated knockdown of p53 expression in making rodent fibroblasts permissive to Ras-induced transformation. These observations indicate that mycoplasma infection plays the role of a p53-suppressing oncogene that cooperates with Ras in cell transformation and suggest that the carcinogenic and mutagenic effects of mycoplasma might be due to inhibition of p53 tumor suppressor function by this common human parasite.

tips para minipreps

http://bitesizebio.com/2008/04/08/5-more-tips-for-dna-gel-extraction/

Quick and dirty

Identificacion de plasmidos con inserto en clonaciones

Reagents:

2x Lysis Buffer:

20% w/v Sucrose
200mM NaOH
120mM KCl
10mM EDTA
0.5% SDS
a pinch of Bromophenol blue
store at -20 degC

Procedure:

There are two ways of performing this screen. If the chances of you having a clones depend on the number of colonies you screen i.e. your “luckiness” factor is abysmal, then obviously you would have a screen a large number of colonies. Then go ahead with the 96 well plate method. If you are the type who usually strikes on the clone in a screen of say 10 to 20 colonies then you could use Eppendorf tubes.

1. Dilute lysis buffer to 1x, at room temperature.
2. Pick the colonies from the transformant plate “patch” to a master plate and transfer remaining to 30 microL of lysis buffer (in Eppendorfs or a 96 well plate)
3. Incubate at 37 degC for 5 to 7 minutes
4. Chill on ice for 5min
5. Spin at maximum speed for 10 minutes ( you would need a compatible centrifuge to spin 96 well plates - which should be sealed with parafilm)
6. Load 10 to 15 microL on the gel (Caution: When trying to load from the lysate make sure you only pick up the supernatant. The gooey mass below (caused by genomic DNA) will stick to your piptte causing pipetting difficulties as well cause the sample to jump out of the agarose lanes. If this happens spin down again for a few minutes and load)
7. Difference in electrophoretic mobility will help you narrow down your clones for mini preps.

De
http://bitesizebio.com/2008/04/02/quick-and-dirty-screening-for-cloned-inserts/

Indispensable

A todos los alumnos
Esta es un grupo de revisiones de tecnicas en Cell. Por favor leanlas, son muy buenas e indispensables
http://www.cellpress.com/misc/page?page=ETBR


p.s. podriamos hacer algunas preguntas en los seminarios

Muy basico, pero util para los que empiezan

Como hacer busquedas en el pubmed?

http://bitesizebio.com/2008/03/05/18-ways-to-improve-your-pubmed-searches/

Controles internos RTPCR

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes

Genome Biology 2002, 3:research0034.1-0034.11doi:10.1186/gb-2002-3-7-research0034

Por favor lean este articulo. Todos aquellos que usen o vayan a usar RT PCR se beneficiaran de este. Valdria la pena estandarizar para cada linea celular
http://genomebiology.com/2002/3/7/research/0034

Autocrine TNFa signaling renders human cancer cells susceptible to Smac-Mimetic Induced Apoptosis

En este paper por medio de una pequeña molécula que estructuralmente se parece al AVPI tenemos un panel de 50 líneas celulares de cáncer de pulmón de células no pequeñas que son resistentes y sensibles a una molécula que mimetiza al AVPI de Smac. (en la imágen no se ve pero pueden ver aquellas lineas sensibles, intertmedias y resistentes, eje de las x linea celular, eje de las y viabilidad medida por niveles de ATP)
Lo interesante de este paper es que que aquellas células que son sensibles a la molécula mimetizadora de AVPI de Smac tienen en común que autócrinamente liberan TNFa a diferencia de las resistentes. La inhibición deTNFa hace resistentes a Smac a las lineas que eran sensibles.

Petersen SL, Wang L, Yalcin-Chin A, Li L, Peyton M, Minna J, Harran P, Wang X.

Autocrine TNFalpha signaling renders human cancer cells susceptible to Smac-mimetic-induced apoptosis.

Cancer Cell. 2007 Nov;12(5):445-56.

PMID: 17996648 [PubMed - indexed for MEDLINE]

BASE DE DATOS DE BASE DE DATOS

Un numero de acceso libre de bases de datos, muy util en Nucleic Acid Research
http://nar.oxfordjournals.org/content/vol36/suppl_1/index.dtl
y, en particular, la tabla de bases de datos
http://nar.oxfordjournals.org/cgi/content/full/gkm1037/DC1/1

clasificacion de cientificos

http://bitesizebio.com/2007/12/11/a-classification-system-for-scientists/