Reportergene

so useless and miserable

2012-01-31T15:18:00.000+01:00

I did not check on the real original publications if the alleged image manipulations spotlighted in this video are really present, or were just fabricated for the video. And I will not do it. However, if the video is really reporting actual manipulations, my main conclusion will be that the peer-review system is going to fail. For my academic CV, publications in peer-reviewed journals are a must, because for academic commissions it is implicit that the peer-review only will provide a reliable body of credibility for my academic research achievements. This video is the proof that for the same investigator, the peer-review system failed at least 24 times.

Where are your cells from?

2012-01-23T17:38:00.000+01:00

During the Christmas pause, I had occasion to spoke with one of my elderly aunts that I probably see once or twice a year. She had always admired my path to pursue biomedical research, but often she had shown to be uncomfortable with the notion that biomedical research implies some lab animal research. So, while eating some killed turkey despite we were not in the need for calories, she argued that now, as the TV says, the experiments can be done without killing, well, poor mice, but, I mean, directly on the cells. I was sipping some Chianti, so I just nodded up and down while she, well... you know... cells. Then, inspired by the wine, I replied back: by chance, do you know where the cells come from? No? Well, from killed mice, mostly pups indeed. My reply was partial, but at least she learnt something that the TV does not say, and I wanted to be partial to give her such a take home message for that day.

The truth is that most cell lines are actually of human origin, mostly, I presume, taken from volunteers. Maybe in the past, it was a little bit different, and I doubt Henrietta Lacks signed any declaration of consent for the use of her carcinoma HeLa cells, but OK, this was the past, you can read this book just in case.

However, if today I order some human hepatocytes, and I can get them for few hundred dollars, I suppose there was some human informed consent before - that no mice could ever sign - so yes, taking apart any argument regarding the scientific value of animal vs cell research, from a pure ethical perspective, I would prefer doing my research on human primary hepatocytes. However, this JCI editorial position read is getting me anxious. And, dear aunt, you better believe cells come from mice, guaranteed! In a few words, the Journal of Clinical Investigation is making an aut aut embargo on Chinese research papers regarding transplantation. Says the Editor in chief:

(we will no longer consider) for publication any submissions pertaining to or containing information about human organ transplantation in China unless there is an attestation that the organ sources are not executed prisoners.

and more:

The only way to guarantee transplant of a liver or heart during the relatively short time period that a transplant tourist is in China is to quickly obtain the requisite medical information from prospective recipients, find matches among them, and then execute a person who is a suitable match.

Tough words! At the beginning, I felt lucky not to work on transplantation, and be relegated in a more naive basic science corner. However, as the hours are passing, I can not stop asking myself: when I place an order, where my human primary hepatocytes come from?

My Xmas gift, the organic iPad made with bugs

2011-12-20T18:04:00.000+01:00

After the December 2009 'Bacteria towing Santa's wagon', this is the ultimate example of life-imitating art. Millions of E. coli bacteria glowing together. Biologists and bioengineers at UC San Diego constructed this remarkable living display by engineering the biological clocks of bacterial cells to fluoresce together like blinking light bulbs. And they synchronized thousands of these blinking bacteria colonies so that millions of them would glow ON and OFF in unison.

The neon bulbs that make up this glowing displays are chambers within tiny microfluidic chips each one containing some 5,000 bacteria. Each bacteria is genetically engineered with fluorescent proteins attached to the biological clocks, and like the single-dots pixels in your computer and tv monitors, each of these microfluidic chambers form what the researchers describes in a recent Nature publication as the ‘biopixel’. The scientists made different versions of these flashing displays: the smallest is done by assembling together 500 biopixels in a microfluidic chip composed of 2.5 millions flashing bacteria, but the bigger display contains already 13,000 biopixels. Do you imagine recharging your iPad with some LB broth?

Original research reference: Prindle et al., A sensing array of radically coupled genetic biopixels. 2011 Nature Advanced Online Publication doi:10.1038/nature10722

Interview at Sciencedaily.com

trends in synthetic biology 2011

2011-11-15T12:59:00.002+01:00

The 2010 has been the year of Venter's synthetic genome. What about 2011, new developments at the horizon?

Synthetic physiology. One goal of synthetic physiology would be to integrate a new genetic function into mammalian cells, i.e., a circuit that can detect a diseased state and then trigger a therapeutic appropriate response. Two studies are pursuing this goal. The group of Martin Fussenegger (Basel, Switzerland) reported their success in developing a light-driven mechanism to control transcription. The 'photoactivation' switch uses melanopsin, a retinal protein that release a calcium surge when exposed to blue light. The researchers engineered it so that the transient of calcium would activate, via calcineurin, the transcription factor NFAT. To prove that the technique has therapeutic potential, they coupled NFAT activation to insulin production in a mouse model of diabetes. When bathing in blue light, the diabetic mice 'switched on' insulin production and prevented the dangerous glycemic excursion consecutive of eating glucose. [Science 332, 1565-1568; 2011]. Interestingly, the trigger of synthetic circuits could be also an endogenous one: the groups of Weiss (Cambridge, MA) and Benenson (Basel, CH) developed together a miRNA-based synthetic logic circuit that identifies and specifically kills HeLa cancer cells. The genetic circuit senses the expression level of a customizable set of endogenous miRNAs and after customizable AND or NOT gating, triggers a customizable cellular response (i.e., apoptosis), opening the way to the design of synthetic circuits for futuristic clinical purposes. [Science 333, 1307-1311; 2011]

A comic appeared on Nature in 2004.

Synthetic life? Venter's cells were not completely synthetic as only the DNA was artificially made. But, is it possible to build a fully synthetic cell? Some progress come from an unexpected approach: synthetic inorganic life. Despite the strong claims of bacteria substituting phosphorous with arsenic in their DNA have been largely criticized, the research of inorganic life continues on different fronts: for instance, the group of Lee Cronin (University of Glasgow) is trying to create self-replicating, evolving, inorganic cells that would essentially be 'alive'. Simple inorganic chemical cells (iCHELLs) can be obtained at the liquid-liquid interface between aqueous solutions of POM (polyoxometalate) clusters and coordination-complex cations with the aid of very simple instrumentation. The researchers placed a drop of cation solution on a glass coverslip and POM solutions were then injected into the droplet using Eppendorf femtotips. The process can be nested resulting in compartmentalization of internal membranes that were found to control the passage of materials and energy, meaning that several chemical processes can be isolated within the same iChell, just like biological cells [Angewandte Chemie Int Ed, 50(44); 10373-76; 2011]. Future steps under investigation are the engineering of iCHELLS able to undergo fission into daughter iCHELLs, just like inorganic life. For the moment, a second in vitro study lead by Laura Martini and Sheref Mansy shows that cell-like systems with riboswitches can be used to control protein expression under fully defined conditions in water-in-oil emulsions [Chem Commun 47; 10734-36; 2011].

packaging madness

2011-10-27T20:54:00.001+02:00

I should say thanks to Nick for having posted yesterday some pictures of Sigma-Aldrich packaging madness. Packaging has a cost and an effect on environment, however, as Nick correctly points, some chemicals shipped by Sigma are dangerous and this might justify some excessive packaging.

This morning I received some chemically-inert stainless steel beads that I'm using to extract RNA with the Tissuelyser, my preferred method. My bench-mate Shawon received some too. Despite we are from the same lab, from the same address, and from the same center of cost, Qiagen sent us two different parcels, therefore doubling the shipping and handling taxes, and doubling CO2 emissions.

The picture shows the amount of stuff required to send 400 x 5mm stainless beads (in the small plastic bag). Our lab paid 352 dollars (subtotal) plus 41 dollars (shipping and handling). This packaging, which costs me 10% of my research budget, offends me every time I make an order, so I've been looking for alternatives. After some common sense thinking, my conclusion is that these are not complex chemicals, but simply inox beads with a diameter of 5 millimeters, nothing more than bearing balls. Next time, saving 60% of my budget, I could get 400 stainless steel bearing balls for 160 dollars.

ELN - Electronic Lab Notebooks

2011-10-11T20:49:00.001+02:00

The opportunity to switch to electronic lab notebooks (ELNs) had been discussed for 2 or perhaps 3 decades without any concrete change. However, the recent boom for tablets, ipads and the new amazon fire costing less than one pipette, is pushing toward a ELN resurrence. We will have ELNs in the lab soon? In the last months, I gave my advisory counseling to some companies considering to enter this market, here I summarize my thoughts about ELNs.

Free! Electronic Lab Notebook: Paperfree from day one, download free tutorial.

For my experience, speaking with senior PIs, the most frequent points against ELNs are:

1) Doubts about patent application eligibility of data recorded electronically (cheating is easier on files than paper, think to edit back a file date vs rewrite a fake classic lab-book with old ink on old paper).

2) Fear to loose the data: files get more easily corrupted than paper and senior PIs are still thinking to backups with the 'floppy-disk' in mind.

My point against current ELN is on the longevity of the file format the data is stored on. In most cases, getting into the market implies inventing a proprietary file format. As it turns out, I have problems to open files created 10-15 years ago because the software is dismissed or not compatible with new operating systems, but I can read a lab book written by my PI 30 year ago. Open formats are preferable to assure that the data will be safe also if the company/product will exit the market.

My positive point is that I can not have such a great benefit in reading an old lab-book because I don't understand the calligraphy or because some points are omitted or spread on different pages/different books. Of course, a search function is not available!

What is the principal ELN function?

In my opinion, the first aim of a lab book is to guarantee the repeatability of the experiment and to summarize the most important results. Summarizing results it is easy on paper: just need to attach a graph or a picture with papertape... But the repeatability is a pain! You have to manually write each detail of the protocol: the batch of the reagent, the concentration, the volume, the number of times you mixed it, the number of times you thawed it. The amount of time you left the tube on the instrument/bench/freezer... You can easily spend a day in writing the details, but Darwin times are over and you are in hurry! Getting your work published today requires 10x more figure panels than in the past, conversely the time to graduate or to get-tenure is the same or less! Today, research scientists are desperately running, so it is natural and unfortunate to evolve a behaviour that skips the parts that are not essential for publication (i.e., keeping a fair notebook). However, the repeatability is then compromised, and the retraction rates are increasing.

What ELNs should do?

An ideal ELN would be so intelligent to auto-fill the details I'm lazy to write down, like a personal assistant that just follows you at the bench and keep notes of your handling without you need to stop. The ideal personal assistant, will have also some memory: ELN, when I used this polymerase last time? It was two weeks ago for the experiment Y. I mean, the ELN should be interrogable, like google is.

The biggest mistake ever done in ELN design

Do not design an ELN as a closed environment that manages lab data. Every lab, every scientists has their own preferences in terms of storing some report on some software: the same graph can be made in excel, open 'excel' alternatives, in graphpad prism, copied to word, copied to powerpoint, distilled to pdf, to jpg etcetera. Scientists are already asked to change working hypothesis/protocols different times a day, dont ask them to change also the 'system' they store the data, they need some sticky habits.

ELN is a personal secretary, nothing more

A good ELN, as a personal secretary, should just be flexible and smart enough to point/link to the different files with a more elastic mind than windows explorer or mac. For instance, I can store my qPCR files in a qPCR folder, this is beautiful if I then ask how many PCR I have done this year, but ugly if I need to retrieve 4 PCRs for the manuscript Y or the 3 PCR paid with a grant X. I can store the PCR for the project Y in the Y folder or the X PCR in the grant X folder. But I'll rapidly mess up stuff. As a clear example, in the last 5 years, my 'Results' folder contains 1905 sub-folders stuffed with 22,187 files for a total of 22 Gigabytes, and I'm not involved directly in any massive sequencing, the core is all about basic western blots, luciferase assays, RT-qPCRs and some microscope picture!

My ideal ELN would be able to assign my preferred file (protocol, raw data file, result file, literature reference, manuscript in preparation, presentation, referee,...) to each of their corresponding folders (project, person, experiment, grant, application), and perhaps keep an historical protection of different file versions. In my mind, more than a software, the ELN should be a sub-operating system able to run as a virtual environment into win, mac, linux and able to mangle my data better than I do, like a personal assistant.

If you are developing some ELN stuff and want my feedback, you can get in touch at info@reportergene.com, confidentiality is guaranteed.

Free! Electronic Lab Notebook: Paperfree from day one, download free tutorial. <

life correlations

2011-09-16T00:32:00.000+02:00

I'm going to the EMBO conference on Nuclear Receptors. Last time I went - four years ago - my wife was pregnant and in the middle of the last talk phoned me because of the labor contractions. My house was 100 km long away, and I come back in time.

Now, my wife is once again pregnant and tomorrow I'm taking the flight. Delivery is expected on February. Hopefully, it should be easier this time.

Why scientists are doing fluorescent pets?

2011-09-12T20:49:00.004+02:00

Q: What is going on? Why scientists are taking our dogs, rabbits, pigs, fishes and now even cats and after some buzz manipulations they transform them into scary fluorescent beasts? What is the point in making the cat to glow?

A: If a laboratory animal glows, it is proof that a genetic manipulation has been done. Our genome (our DNA) is too small to hold it down with mini-forceps and to model it like nakiplast: if a scientists wants to modify a genome, he will use more indirect methods requiring at the end some proof that the DNA has been actually changed. The set of methodologies generally called 'transgenesis' refers to the modification of the genome by insertion of a new gene that normally gives to the host genome a new function. Since most transgenic techniques are species-specific - if a technique works for rats maybe does not works for frogs - scientists are urged do develop new transgenic techniques to study the major number of species* from a genomic point of view. Usually, for the proof of actual transgenesis, scientists are introducing first a 'proof' gene, aka a 'reporter gene' like the one that gives to the animal all the instructions to build the green fluorescent protein. Therefore, if your cat glows in the dark, it is because in its genome there is a new gene, the GFP one.

Transgenic fluorescent kitty

A fluorescent pet, like the one in the picture published today by Dr Poeschla in Nature Methods, is the proof of concept that a new transgenesis technique works for cats. In addition, because GFP is visible at the exterior of the animal and because it is possible now to couple the expression of a reporter gene with any other 'molecular gear' reporting any physiological activity, this technological advance is the promise that in future biomedical research will be conducted from an exterior point of view, without the need to kill the laboratory animal. Today, this is only a promise, and it will probably require decades, because in science every step need a 'proof', but it's where I'm playing hard to get it done.

In the while, we will still write and read these 'bypass' declarations in the reported research.

Three male and two female transgenic cats, named TgCat1–5, were born by spontaneous vaginal deliveries at term and all five were transgenic. TgCat1 (male), TgCat2 (male) and TgCat3 (female) survived, whereas the fourth and fifth cats died perinatally from obstetrical complications. [...] TgCat4 was born after an uncomplicated singleton pregnancy at a normal gestation time (65 d). It was morphologically normal but died during or shortly after parturition from an apparent obstetrical accident involving aspiration, although a precise cause could not be determined at autopsy. This cat provided the opportunity to study all tissues. [...] and immunoblotting revealed abundant GFP expression in all tissues tested: brain, spinal cord, heart, spleen, skin, muscle, liver, kidney, small intestine and stomach.

-------/ Original citation: /-----------------
Pimprapar Wongsrikeao, Dyana Saenz, Tommy Rinkoski, Takeshige Otoi and Eric Poeschla
Antiviral restriction factor transgenesis in the domestic cat
Nature Methods (2011) doi:10.1038/nmeth.1703

* we need to study the major number of species because different animals have different similarities to human biology: for instance our brain is more similar to the brain of a cat than the mouse brain.

This is important, go and read

2011-09-06T21:52:00.000+02:00

Jonathan has just made a call to bloggers and authors of peer-reviewed research manuscripts to get used in aggregating publications with online comments. I find this important because we can achieve a second-layer that better connects scientific community and, as a consequence, facilitates meritocracy. Two years ago I wrote something about it in: does a blogger influence IF.

So, go to read Jonathan's call.

recursive damnation of the researcher

2011-08-31T21:00:00.002+02:00

In three blockquotes and one blog post

1) Painful question:
By sitting all day long to write engaging specific aims. I'm perpetuating a dead science model?

It is reasonable to assume that the best researchers end up being PIs, eventually. Yet in the current system, all their time is consumed in writing grant proposals, and managing their research groups. This is a waste of their potential: they are supposed to be the best researchers available to the community yet they cannot perform any actual bench work!

Michel Valstar on the problem of monetarising academic science

2) Painful argument:
Yes but if I pass my time with the pipet, I'll never get the manuscript written!

Academic publishing is an odd system — the authors are not paid for their writing, nor are the peer reviewers (they’re just more unpaid academics), and in some fields even the journal editors are unpaid. Sometimes the authors must even pay the publishers. And yet scientific publications are some of the most outrageously expensive pieces of literature you can buy…. As far as I can tell, the money paid for access today serves little significant purpose except to perpetuate dead business models.

Mathew Ingram waiting for the death of academic publishing

3) Painful swearing:
OK, but if I don't publish twice in a year I'll never get the f@#€d tenure!

Research institutions pursuing high-risk research may have to rethink evaluation timelines and the criteria for judging and rewarding performance. Achieving the goals of a transformative research program can take longer than those of an incremental program designed in a more linear way; transformative research can have many false starts and thus a reduced publication rate.

Alan Leshner for increasing the diversity of the scientific human resource pool.

4) Recursive damnation of the researcher:
Cool, let's write a blog post.

Go back to question 1.

Where I try a synthetic plasmid

2011-08-17T17:07:00.004+02:00

DNA synthesis can be cheap, but don't get fooled by the ads: be aggressive and make them negotiating against themselves.

I had this collateral project in mind for a while, but to realize it, I should have taken a serious cloning effort, which was asking energies not available at the moment. For my scope, I needed to have in the same plasmid a reporter gene, a protein fusion, some responsive elements made from very repetitive elements, different polyA and stop codons to minimize the interference of the different ORFs, a multiple cloning site to subclone protein mojeties in frame with a second protein. Moreover, I was flanking my construct with selected restriction sites for subsequent adenovirus production, and I had to check that no others restriction sites were present in the whole plasmid. A nightmare of 3000 base pairs.

Not surprisingly, I postponed this low-priority project for almost two years. Then, at a certain point, comes Craig Venter, who used gene synthesis to make up an entire genome. Ah-ha! I searched in google "Gene Synthesis" and found encouraging ads around $0.29/bp (check now for updates).

I had never used GS before, and I was lazy to ask quotes and procedures to ~~piranha~~ customer services, but the MrGene site made me happy. It just required to paste my sequence and it was giving a quote in real-time. So, I assembled my sequence in Word (don't scream, it was my best choice to annotate with colors the different gene parts, do you have alternatives?), paste it into MrGene and get a pdf quote back. The site informed me that the quote was higher than the standard because of the lenght (3000 bp) and the repetitive sequences, but was fair around $0.65/bp. For me it was fine, I just needed a few weeks to find the money (~ $2000), to discuss adenovirus production with collaborators and I was ready to start.

When I went back, MrGene was closed as it was acquired by Invitrogen. I asked again a new quote: ~ $3000! ($1/bp) I was out of the budget! I contacted my local agent, who promised to do its best, he put me in contact with another guy, who put me in contact with the guys at Gene Art (?) that actually were managing the stuff for Invitrogen. However, they asked me to send again the sequence because:

From a technical point, we would need to take the two projects from the portal and make it all a manual order, as there is basically no flexibility in the automated ordering online portal. So it would be great if you could send me the sequences (including all details) again via email and then I can see how we can arrange the two projects to match your requirements

Apparently, nobody was able to explain me how the hell the same quote was increased by 50% just because of a change in the brand, but they gave me a stupid code that I could have used to obtain a undefined 'discount' for other, more complex projects. However, my project was complex enough to have my sequence lost in their server.

At this point I was in the jungle, therefore I started asking quotes to several companies, putting one against the other, and the results of this trip in negotiation-land are summarized in the graph below.

Six weeks later, I saved the budged receiving the plasmid from Biobasic Canada. I had 4 ug of my synthetic plasmid safely shipped in a liophylized form to my collaborator for adenovirus production, the sequence was 100% correct. The project is running without any cloning from my part. Today, Genscript and DNA 2.0 are spamming my inbox with unsolicited newsletters about their services. The Invitrogen code for the discount is P754233, in case you trust it.

-----------------------------------------------------------------------

Disclaimer: this post truly reflects my experience and the quotes I received. I don't have any conflict of interest to disclose. You may get better quotes for smaller sequences. Next time, you might offer better quotes to me if you want to perform better in my graphs, period.

Friday corollary: homeopathy

2011-08-05T15:23:00.001+02:00

If homeopathy worked...

...the Olympic Committee would have already banned it

(with apologies to the conditional)

A tool to write rapidly your next Science paper

2011-07-27T22:25:00.000+02:00

Google Scribe might help you in writing better manuscripts.

Let face it, most scientist are not English mother-tongue. Me neither. It is important for me to write clearly when I want to communicate an idea, however it is still painful to build some key phrases as the construction that I have in mind in Italian, does not fit with English. I'm obliged to bend my argumentation to fulfill English rules, but while doing so, my writing loses immediacy, because my English is not smart. Not yet.

Here comes Google Scribe. Apparently, when you write, Scribe suggests you relevant words, thanks to a comparative engine that is based on a correct English corpus. This is good, I mean, I'm not obliged to follow Scribe's suggestions, but having them in real time, allows me to have the pulse about my distance from average English.

If you didn't try yet Google Scribe, it is worth a minute of your time. Just start typing, if you accept a suggestion, a space bar stroke will fix it and you will continue typing the next following word. If you think rapidly, this is really going to increase your writing rate.

Now comes the question: how does Scribe prioritizes its suggestions? I think it could be useful to tell it what you are going to write: a love letter? a scientific paper? a novel? Because Google Scholar and Google Books are plenty of full-text corpora, it could be good if you could adapt the suggestions based on the appropriate corpus. So, I added this idea in Scribe's feedback poll box. If you like it, you can vote it.

IMPORTANT: cell culture users

2011-07-13T17:46:00.002+02:00

[found this in my inbox this morning, not able to stop laughing]

Dear all,

Please note that the electricity plug of the pipetboy HAS NOT to be plugged in the place in which we enter the pipet!!! (we had this problem in the 2 hoods in the cell-line room). It will not help recharging the battery in any way...

The electricity plug is located at the bottom of the part we put in the hand.

Regards,
Xxxxx xxxx

new engaging search

2011-05-27T11:44:00.000+02:00

Today I implement Apture, a new way to search for relevant content. Simply highlight an interesting phrase on reportergene, (any phrase, just pick one!), and you will see a bar loading entitled "Learn More".

In a second you will be presented with a variety of articles and videos related to that topic.

If you find it useful, let me know in the comments.

google fails

2011-05-25T20:07:00.000+02:00

Dear Sergey,

I admit it, I have a bad keyboard and a scarce technical ability in typing. Most of the time, I do not write at the first shoot the word I want to write. And when it occurs, it turns out to be the wrong spelling, sorry. For me, that's fine, I'm not ashamed of it. Actually I like it: I think too fast, and slowing down helps to focus my toughts. (I took me three times before writing toughts correctly).

Recently, it happened to use your software based on your publication "The Anatomy of a Large-Scale Hypertextual Web Search Engine" submitted to WWW7. Well, concerning this software I appreciate your tolerance, and your kind attention to my spelling. Thanks for saying: "maybe you were looking for..." Very polite, thanks. That was fine. Now let's talk about 'scholar' version of this search engine. Here is different. I can spend minutes before searching the exact terms I need for. I less appreciate when you change the results of my search just because you know I'm wrong. No, you don't.

If I look for 'pestrogen' I was not looking for oestrogen. Don't put oestrogen in my results, or you will saturate my patience. When I search 'heme' + 'my favourite gene' I'm not looking for a theme on my favourite gene. That last example returned 4 wrong search results out of the first 10. That's 40% of false positives. From an academic man, to an academic man, with such a bad result, I'm afraid you will never get your PhD. And, google will never get enough impact factor. :-)

best job ever

2011-04-08T11:16:00.000+02:00

I'm analyzing my data, and I have a long list of numbers. It is the magic moment when I'm aware that technically, the experiment worked fine, but yet I don't have idea of the differences between groups. And while I'm reshaping the data to make a meaningful table, I feel my heartbeat increasing, and adrenal glands secreting exciting hormones in my bloodstream. In a couple of minutes I will know whether it will be a good day or not. Anachronistically, I still prefer juggling manually with data and copy-paste excel tables rather than scripting with R. It's a low, exciting, data uncluttering, and I honor it with the time it deserves. Definitively, research is the best job ever.

DIY yourself transgenic mice

2011-03-28T10:25:00.000+02:00

Although I'm not conducting active research on it, a question that fascinates me is: it is possible to get a transgenic model in a manner as simply as making cell transfection? My feeling is that it would be simpler to follow a sperm approach, by 'trasfecting' sperm and then use it for artificial insemination. In a previous post, I wrote about some guys electroporating the testis (ugly!). Now, a letter in Nature open a new perspective, although it is not discussed in the paper. Indeed, Sato et al. took fresh or frozen testes from 2- or 3-day-old GFP transgenic mice and grew some organ slices at 34 °C for about a month in a agarose petri dish with media (alpha-MEM) containing KnockOut serum replacement (KSR), B-27, AlbuMAX, HGF, Activin A, FSH, testosterone, BMP-4 and BMP-7 recombinant proteins, and bovine pituitary extracts. Actually, for the study they took advantage of two different GFP reporter mice as testes donor: Gsg2-GFP and Act-GFP. In vitro, the spermatogenesis looked fine both in terms of meiosis (Gsg2-GFP imaging) and in terms of generation of haploid cells (Act-GFP imaging) and eventually mature and functional sperm was produced. Sperm could be extracted, artificially inseminated in female mice, and healthy pups were generated. I'm sure the technique will further improve and diffuse, as it might be an attractive path for diagnostic and therapeutic techniques for male infertility. By the way, I think having the stem-to-sperm cell in the organ culture to be stably transfected with a foreign gene, could be quite simple to obtain. To make a transgenic mouse you would just need to by some spermfectamine. As Bill is saying, a transgenic in every bench.

--- ---/ link to primary source /--- ---
Sato, T., Katagiri, K., Gohbara, A., Inoue, K., Ogonuki, N., Ogura, A., Kubota, Y., & Ogawa, T. (2011). In vitro production of functional sperm in cultured neonatal mouse testes Nature, 471 (7339), 504-507 DOI: 10.1038/nature09850

A life form violates the copyright

2011-03-25T18:19:00.000+01:00

The cobalt blue colonies of Mycoplasma mycoides JCVI-syn1.0 are facing the same problem google books is experiencing with copyright law. So sad.

In order to distinguish their synthetic DNA from that naturally present in the bacterium, Venter’s team coded several famous quotes into their DNA, including one from James Joyce’s A Portrait of the Artist of a Young Man: “To live, to err, to fall, to triumph, to recreate life out of life.” After announcing their work, Venter explained, his team received a cease and desist letter from Joyce’s estate, saying that he’d used the Irish writer’s work without permission. ”We thought it fell under fair use,” said Venter.

via: Forbes

trends in biomarker mice [March 2011]

2011-03-21T11:52:00.003+01:00

Liver cancer self-reporting mouse. A cassette encoding Tk-ires-Luc was knocked right behind the Afp promoter. Alpha-fetoprotein (Afp) gene is expressed in fetal liver, silenced soon after birth, and highly re-expressed in hepatocellular carcinomas (HCC). Therefore, the novel reporter mouse model enables dual modality imaging of hepatocarcinogenesis in vivo by positron emission tomography (reporter Tk) and bioluminescence imaging (reporter luciferase). The model spotlights cancer development before neoplastic lesions are evident by histological examination. [Lu et al., Journal of Hepatology 2011 in press]
Kidney injury self-reporting mouse. A cassette encoding a double fusion Luc2-mCherry was knocked right behind the Ngal promoter. Neutrophil gelatinase-associated lipocalin (Ngal) gene was identified in ischemic kidneys and its biomarker potential confirmed then in kidneys injuried during hypoxia, drug toxicity and bacterial infections. As Ngal is not expressed in normal kidney, the new biomarker mouse model enables space-temporal imaging of kidney damage in vivo by bioluminescence imaging (reporter Luc2) and fluorescence imaging (reporter m-Cherry). In the model, the reporter signal correlates with the intensity of the injury and seems more rapid and sensitive than renal biomarkers like serum creatinine. [Paragas et al., Nature Medicine, February 2011]
Constant gardener, wanna make a biomarker plant? Here a short protocol describing step-by-step procedures to biolistically introduce fluorescent reporter proteins into lily, tobacco and Arabidopsis pollen grains. [Wang and Jiang, Nature Protocols, March 2011]

I cannot live without it

2011-03-17T18:12:00.000+01:00

TissueLyser, I love you.

Fluorescent dogs 2.0 (with switch ON/OFF)

2011-03-08T18:12:00.001+01:00

Do you remember Ruppy, the red fluorescent puppy? The same team just described a new transgenic dog, this time with inducible fluorescence. The principle is always the TetON/TetOFF. According to the paper publised on Genesis, compared to mice, in dogs you need 10 less the dose of doxycycline (per chilogram) to switch ON the canine GFP, as determined by analyzing GFP expression in several organs. It is less clear how they took the organs: in the previous paper they said that one dog died due to chronic bronchopneumonia, but this time they skip commenting on.

This study does not make dogs easier to spot at night, so fewer will get hit by cars, but illustrates the possibility to obtain conditional expression of a given gene in dogs, after administration of the drug doxycicline (the switch ON). This could be helpful for the generation of dog models of dominant genetic diseases (i.e., by inserting the disease gene). Honestly, I have some problems to think at dogs as 'research tools'. However, the proof of technology described in this paper could be useful on other fields. I'm thinking at the interface between man and dog relations, what about dogs with super-noses to better alert avalanche victims, gas spills or truffles (i.e., using olfactory receptor genes from other species that are less social like fruitflies)? This could made biomedical research useful for the society on branches other than the healthcare.

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Kim, M., Oh, H., Park, J., Kim, G., Hong, S., Jang, G., Kwon, M., Koo, B., Kim, T., Kang, S., Ra, J., Ko, C., & Lee, B. (2011). Generation of transgenic dogs that conditionally express green fluorescent protein genesis DOI: 10.1002/dvg.20737

trends in transgenesis [February 2011]

2011-03-04T11:38:00.001+01:00

Easier Rat IVF. In vitro fertilization in rats is inconvenient and rarely adopted due to long and consequent working hours. A new protocol completing in 'working hours' is now available to facilitate embryo banking. [Aoto et al., Transgenic Research, February 2011].
A new Cre deleter. To efficiently obtain total deletion of your floxed alleles, it is now available a new total deleter obtained knocking Cre recombinase into the Stella locus. Stella (aka PGC7 or Dppa3) promoter is expressed as early as in the two-cells stage embryo till embryonic day 15 in the mouse. [Liu et al., Genesis, February 2011].
Genetic swapping. You plan to make a transgenic mouse to test a protein X? You might start with a X-GAL4 construct to test the ability of protein X to modulate GAL4-dependent UAS-luciferase expression. But then? With the new integrase swappable in vivo targeting element (InSITE) you could simply repurpose your transgenic by swapping in vivo the GAL4 sequence with any other one (i.e., IFP to localize protein X by infrared imaging; GST to pulldown X and make interactome proteomics). It is just matter to make some breedings. [Gohl et al., Nature Methods, February 2011]

cartilage transgenic mouse

2011-02-28T11:23:00.001+01:00

Cartilage is the flexible connective tissue between our bones, and it is mainly based of cells called chondroblasts that secrete a gel-matrix made of collagen proteins. If cartilage maturation is disrupted, diseases may develop like the painful osteoarthritis and other chondrodystrophies. Therefore, it is important to understand the dynamics of cartilage formation and maturation, and a new reporter mouse made with classic pronuclear injection of the linearized product of a bacterial recombination of a BAC clone can help on this.

Collagen can be seen by histological staining (i.e., Tricrome Masson),
however this techniques is poor in rendering the space-temporal
development of cartilage maturation. By contrast, fluorescent imaging
with reporter mice identifies spots of cartilage maturation without
need to kill animals for dissection diagrams.

During cartilage maturation, the chondrocytes produce different types of collagen. Collagen, type 2, alpha 1 (Col2a1) is abundantly expressed in immature chondrocytes, but then is down regulated during maturation. In contrast, Collagen, type 10, alpha 1 (Col10a1) expression is absent in immature chondrocytes, but becomes highly expressed in mature hypertrophic chondrocytes. By following in real-time the expression of the two collagens in the developing bone, one could have an idea of the dynamics of cartilage maturation. This approach has been taken by Peter Maye and colleagues from the School of Dental Medicine (University of Connecticut) who developed a double transgenic reporter mouse in which collagen 2a1 promoter drives the expression of a cyan fluorescent protein (Col2a1-ECFP), and collagen 10a1 promoter drives the expression of a red fluorescent protein (Col10a1-mCherry). In a report published on Genesis, the authors illustrate how the fluorescence of the two reporters can be spectrally discriminated in vivo to efficacely monitor cartilage development. In the picture of a developing foetal mouse, long bones are maturing with extremities being the main center of ossification, while in the middle of the two extremities, cartilage is already differentiated. Conversely, the ribs are maturing following a dorsoventral pattern. Increasing the microscope magnification, it is possible to monitor single cartilage cells and identify the exact time at which an intermediate region switches its differentiation program.

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Maye, P., Fu, Y., Butler, D., Chokalingam, K., Liu, Y., Florer, J., Stover, M., Wenstrup, R., Jiang, X., Gooch, C., & Rowe, D. (2011). Generation and characterization of Col10a1-mCherry reporter mice genesis DOI: 10.1002/dvg.20733

ceci n'est pas un ppar

2011-02-25T14:18:00.001+01:00

You can have a plant, a dog, a cat, even a human partner. You can mix your DNA with him/her and have some children to take care then. But. But in the lab, you have another family to take care. A family of pet molecules. And you live with them for years, you bring them in your mind everywhere you go, you nourish them, you follow their paths. You hate their displacements from your intended logic grid and then you still do love their idiosyncrasies. In the last ten years, I spent an insane amount of time with my pet molecules. Here are two of them: PPAR and RXR, two nuclear receptors. Or at least, here it is what is closer to my mental representation of them. (You can play with your mouse, it is interactive).

Being our physical and logical interactions separated by orders of magnitude, I never saw them. I never touched them. I based my relationship on some traces they put on my path and I call it scientific research, but sometimes it looks like more interrogating a divine dimension. I got completely fooled this week, thanks ppar is friday.

The widget for posting tridimensional molecules kindly available here.