optogenetic control of intracellular signaling

ResearchBlogging.orgPharmacokinetics and pharmacodynamics should include in future opto-kinetic and opto-dynamic disciplines. At Stanford, Raag Airan and colleagues developed opsin-receptor chimaeras (the optoXR family) as a new class of retinal-based tools. In a Nature letter, they show that the class of OptoXRs can be functionally expressed in vivo, to permit differential photoactivable control of intracellular cascades with significant impact on the phenotype (i.e., behavior when light was targeted in brain via 200um optical fibers). A green light stimulation (~500 nm, 7 mW/mm2) was sufficient to increase intracellular concentrations of (cAMP, IP3, Ca2+) at levels comparable to those induced by pharmacological stimulation. The use of light, instead of drugs, to modulate network physiology, would be of substantial interest given the versatility of optics to precisely define relevant stimulation times with great control of pulsatile versus tonic modulation. This is also of major concern for synthethic biology, given the wide portfolio of photo-proteins and appropriate light-responsive elements.

Airan, R., Thompson, K., Fenno, L., Bernstein, H., & Deisseroth, K. (2009). Temporally precise in vivo control of intracellular signalling Nature DOI: 10.1038/nature07926

Predicting enhancer activity [guest post]

ResearchBlogging.orgLess than 2% of genomic DNA codes for protein. The remaining noncoding portions have been dismissively referred to as junk. Junk implies that because the DNA doesn’t code for proteins, it isn’t functional. In recent years, researchers showed that so-called junk DNA contains regulatory regions, promoters and enhancers that regulate gene expression. Identifying and cloning a gene is one thing, but knowing when and where it’s expressed is crucial to understand how organisms develop and function. Identifying regulatory regions, however, has been a challenge. Promoters tend to be located adjacent to the genes they control, but enhancers are scattered throughout the genome, sometimes 1 million bases of DNA away from the gene they regulate.
Axel Visel and his colleagues found a way to identify when enhancers are regulating genes. A protein called p300, expressed throughout the body, is found in many enhancer-associated protein complexes. p300 is also required for embryonic development, a crucial time when activated genes are literally building the body. Visel dissected forebrain, midbrain and limb tissue from more than 150 mouse embryos, cross-linked the DNA and protein, and digested the DNA (pieces of DNA bound to protein are protected). Using antibodies, Visel purified only those pieces of DNA bound to p300 and then sequenced that DNA and identified it as a possible enhancer by alignment to the mouse genome. This technique, called chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq), is not new, but using p300 as the bait was a clever twist.
To confirm these regions of DNA regulate gene expression in vivo, Visel identified orthologous regions from human DNA. Candidate enhancers, average size 2.4 kb, were cloned upstream of mouse minimal hsp68 promoter and lacZ, a reporter vector used previously by this and other groups. Candidate enhancers were not cloned in any particular orientation. Vectors were injected into fertilized mouse eggs, the eggs were implanted and at embryonic day 11.5 embryos were harvested for whole-mount X-gal staining. Only similar staining patterns observed in three different embryos (representing three independent transgene integrations) were considered valid. If ChIP-seq identified an enhancer active in the limb but not in the brain, then the human orthologue of the enhancer should turn only the mouse’s limbs blue. In most cases, that’s what happened. (See photo.)

Overall, 87% (75 out of 86) of the enhancers produced expression patterns in mice that agreed with the ChIP-seq results – a tremendous improvement over the same researchers’ previous prediction method (47%, 246 out of 528), in which enhancers were identified based on evolutionary conservation and tested using the same reporter assay in transgenic mice. The p300 ChiP-seq method is especially good because it’s large-scale, enabling scientists to study thousands of enhancers throughout the genome from any tissue during any time in an animal’s life. Visel and colleagues identified thousands of enhancers active in the brain and limbs of mouse embryos and verified 75 using transgenic F0 embryos. Using this technique, future studies can identify in vivo enhancers from additional anatomic regions, embryonic (or adult) stages, and from mouse models of human disease.
As my graduate adviser used to say, if you put junk in, you get junk out. Clearly junk DNA is anything but.

Visel, A., Blow, M., Li, Z., Zhang, T., Akiyama, J., Holt, A., Plajzer-Frick, I., Shoukry, M., Wright, C., Chen, F., Afzal, V., Ren, B., Rubin, E., & Pennacchio, L. (2009). ChIP-seq accurately predicts tissue-specific activity of enhancers Nature, 457 (7231), 854-858 DOI: 10.1038/nature07730

Daniel Gorelick is a neuroscientist who is currently taking a year off from his postdoctoral fellowship to serve as a AAAS Science & Technology Policy fellow in the U.S. Department of State. He writes the Science Planet blog and covers science and technology for www.America.gov, a State Department Web site. E-mail: scienceblog@state.gov

define gene

ResearchBlogging.orgIf we want to come grips with new "reporter genes" we need to operationally know what is a gene. Giving a gene definition is a complex trivial task. For our purposes, wikipedia is not exactly so strictly operational:
A gene is the basic unit of heredity in a living organism.
More helpful is probably the definition recently proposed by Graziano Pesole:
A gene is a discrete genomic region whose transcription is regulated by one or more promoters and distal regulatory elements and which contains the information for the synthesis of functional proteins or non-coding RNAs, related by the sharing of a portion of genetic information at the level of the ultimate products (proteins or RNAs)
The take-home lesson is that a genetically-encoded assay should not necessarily rely on expressed proteins.

PESOLE, G. (2008). What is a gene? An updated operational definition Gene, 417 (1-2), 1-4 DOI: 10.1016/j.gene.2008.03.010

cook-it-yourself transgenic pets

Via Biotechniques, I found such interesting MIT-tube, with several videos.
According to Natalie Kuldell, computer engineering advanced quickly that genetic engineering because technology was available to amateurs.
Building biological components and streamlining processes is difficult in biology, because biosystems are complex, and unpredictable. Can amateurs working with tupperware, thermometers and genetic engineering in the kitchen discover something remarkable doing their biology at home?

Previous similar posts: rebuilding a dino, have fun with luciferase, banana imaging.