How to improve reporter activity

Got a weak promoter in exam and your reporter signal is very close to background? Welcome to the club. Discovery companies tried to approach this problem designing for you more stable reporters (that accumulate over time), but they ask you to sacrifice any dynamic curiosity about what is going to happen over time. So what you can do?
In 1988 some viral sequences were described to initiate ribosome binding and translation in a cap-independent manner, they were named IRES (Internal Ribosome Entry Site) and they opened the way for bicistronic vectors. So, lot of scientist make construct like this:

PROMOTER-GENE1-IRES-GENE2-polyA

One single promoter drive a double transcript containing two messengers; then the IRES is supposed to drive to the ribosome the GENE2 alone with an efficiency ranged between 20% and 50% of the cap-dependent GENE1. This constitute the major disadvantage of bicistronic reporter.

When Bouabe and colleagues from Munich University comes to the club of weak promoter, they wondered: "Why don't put another copy of the same reporter after the IRES?".

PROMOTER-REPORTER-IRES-REPORTER-polyA

Such approach is described in Nucleic Acid Research and constitutes a simple technology that allows improvement of reporter gene activity using multiple IRES-Reporter linked in tandem. Basically IRES allow the same weak promoter to express more reporter without interfering with its transcriptional dynamics. Fluorescent proteins like EGFP (that to date, need to be at least 10,000-100,000 molecules/cell in order to be visualized) are probably the reporter that would benefit more of such improvement, without the need to touch their stability.

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Bouabe, H., Fassler, R., & Heesemann, J. (2008). Improvement of reporter activity by IRES-mediated polycistronic reporter system Nucleic Acids Research, 36 (5) DOI: 10.1093/nar/gkm1119

New address

After 6 months, 34 posts, and up to 2000 pageviews, Reportergene got his own domain at www.reportergene.com

It's a milestone, thanks to everyone of the 56 countries that read Reportergene until now.

Multi-reader from Promega

Promega Corporation just released the GloMax-multidetection system: with multi-mode capability, such luminometer can read also fluorescence and absorbance in 96-well plates.

1. Luminescence Module: An advanced photon-counting photomultiplier tube (PMT) can detect as little as 3 × 10^–21 moles of luciferase, covering a dynamic range over 8 logs. A dual masking system minimizes well-to-well cross-talk.
2. Fluorescence Module: Application-optimized Optical Kits simplify fluorescence operation, long-lived LED-based excitation lights minimize maintenance and variability in intensity. The UV, Blue, Green and Red Optical Kits are included with the Fluorescence Detection Module. An AFC Optical Kit is available as an accessory.
3. Absorbance Module: A 6-position filter wheel with 2 open positions. An LED-based visible spectrum light source minimizes maintenance and variability. Filters for reading 450, 560, 600 and 750nm are included. A 490nm filter is available as an accessory.

According to the company, the system is applicable for cancer research, cell biology and drug screening. With a simple touch-screen interface, the system operates as an independent work station without the need for a dedicated computer. Scientists can transfer data to a computer at any time via the system's USB port.

Promega Corporation | www.promega.com

Iranian firefly with red-shift

There are some Iranian scientists that, instead of pursuing nuclear programs, pursue optical ways. Obviously I'm talking about reporter genes, what else? Tafreshi and colleagues, from Tarbiat Modares University in Tehran, recently described in the Biochemical Journal, a site-directed mutagenesis (His245Asn) of luciferase from Iranian firefly (Lampyris turkestanicus). According to the authors, the mutant luciferase holds a promising future for in vivo imaging, because this mutation retain an astonishing activity (76% of wt) compared to other red luciferases like firefly S284T (26% of wt). Red-shifted reporters (as mentioned for the fluorescent katushka some months ago) are very appealing for in vivo applications, because biological tissues and hemoglobin absorb preferentially green-yellow light. Red light is not absorbed, as shown by my hand in front of the lamp.

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Tafreshi, N., Sadeghizadeh, M., Emamzadeh, R., Ranjbar, B., Naderi-Manesh, H., & Hosseinkhani, S. (2008). Site-directed mutagenesis of firefly luciferase: implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases Biochemical Journal, 412 (1) DOI: 10.1042/BJ20070733

Luciferase reports Maternal Cell Chimerism

Are you a chimera? If in your body, some cells belong to your mother, you are a chimera of cells. Better, you are a living example of maternal cell microchimerism (MCM). In humans, maternal cells are present in the affected tissues of children with inflammatory myopathy, scleroderma, and neonatal lupus. The role of MCM in pregnancy and disease pathogenesis remains to be elucidated, and first of all, there is a need for background information (how many cells? how many tissues? how often?).

Su and colleagues from Tufts-New England Medical Center exploited reporter gene technology to adress some basic question: they bred female CMV-luciferase mice (from Xenogen) to wild type (WT) males. The WT offspring were sacrificed at various postnatal ages, multiple organs where microdissected and DNA was extracted for real-time PCR amplification of Luc transgene as a marker for maternally derived cells. Sensitivity was 1 to 2 transgenic cells per 100,000 WT cells. MCM was noted in 85% of mice and 45% of tissues assayed. Basically the authors draw a baseline for future mechanistic studies of MCM using qPCR amplification of a maternal genomic marker (the reporter). But what about in vivo imaging claimed in the title? They used such costly tecnique just for genotyping/phenotyping purposes (discarding luciferase-positive mice), this is a non-sense! Why not using PCR (all the paper is centered on PCR)?

Su, E.C., Johnson, K.L., Tighiouart, H., Bianchi, D.W. (2008). Murine Maternal Cell Microchimerism: Analysis Using Real-Time PCR and In Vivo Imaging. Biology of Reproduction DOI: 10.1095/biolreprod.107.063305

disillusion #1 - I'm a referee

Prologue: It happened! I'm a referee! A minor journal asked me to review a review. It was my first time!

Action: Unfortunately my university doesn't provide access to that journal. No problem, I expected that, as a referee, with my ID and password I would able to get a sharp idea of the journal editorial cut. Naive man. I asked why not. Reply:

"We appreciate your email regarding this issue. However, we do not provide online access to our reviewers".

Conclusion: I'm a voluntary reviewer for free, I cannot read the paper I reviewed, I don't know the editorial cut of the journal I review, I will not cite any paper from such journal in my future papers (I can not read them!), but I CAN pump my resume with "I'm reviewer for that Journal".

Epilogue: my colleagues said: "of course, you have only to make indications about the scientific level". I don't disagree, but I'm still un-satisfied. Why? Naive man!

You may also read: disillusion #2 - presenting a poster

Blueprint - A biotech thriller

"I don't like the word clone. I prefer to call myself blueprint."
Imagination based on reality forms a crucial part of a scientist's life. It means uncovering masses of details and then juggling creatively with the results. Roche aims to offer every month inspiration from a selected biotech thriller giving a chance to win one of 50 DVDs of the respective biotech thriller. The February Imaginality Thriller is Blueprint by Charlotte Kerner and it's based on reproductive cloning.
"Blueprint" is the biography of the adult clone Siri Sellin: The highly gifted, but incurably ill 30-year old Iris Sellin decides to have herself cloned, bring this clone, her "twin", into the world and thereby prolong her life through this identical child. "Blueprint" presents the devastating psychological consequences for the clone Siri: she is not unique and her clone is her mother. Only with the death of Iris is she finally able to find her own identity.