Site-specific recombinases have, for the past 20 years, been one of the most powerful tools in studying the functions of all sorts of genes. Most widely used as the Cre/lox-based system for inserting or deleting genes in mice, transgenic analyses have told us volumes about animal development1. For instance, mouse genetic knockouts are routinely the most convincing method available to determine the most vital function of a given gene product.
In some tissues however, imaging of intricate cellular structures is very difficult. In the brain, for instance, it can be difficult to distinguish one cell from the next in a synaptic circuit, and even more challenging to render a map of neural connections that include divergence and convergence of synaptic relays between many neurons. Fluorescent labels and other staining techniques have been used, to limited effect, but ‘connectomic’ maps of many or all neurons has been beyond the reach of available methods.
Enter Jeff Lichtman and colleagues2. Their paper in this week’s Nature offers an innovative approach to the problem: Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. The method, which they call a ‘Brainbow’ system, takes the existing Cre-lox transgenic method and turns it into a combinatorial expression approach. Their approach actually involved two genetic strategies used in tandem, for stochastic expression of multiple fluorescent proteins (XFP’s) from a single transgene. Brainbow-1 used Cre-mediated excision between pairs of incompatible lox sites, alternated to create mutually exclusive recombination events. Brainbow-2 used Cre to invert DNA segments delimited by loxP sites in opposite orientation, positioned in tandem to generate several recombination outcomes. The differential expression of multiple copies of these constructs generated XFP mixtures, allowing the labelling of individual neurons and glia with as many as 90 distinguishable colors.
The result is a pretty impressive proof-of-concept demonstration. Figure 5 (above) is just amazing, depicting cerebellar circuit tracing and color analysis.
Neuroscientists have mapped the brains regions fairly well on the anatomical scale, and shown neural processes on the cellular scale. The inference has long been that when massive amounts of neural circuitry come into play, then new properties emerge, such as consciousness. Brain imaging techniques such as functional MRI have shown us where consciousness is manifested in the brain, but not how. Maybe this new technique will help us to resolve how different neural circuitries influence consciousness*.
* = Livet et al. make no mention of this speculation, that is all me.
Branda CS, Symecki SM. (2004) Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev. Cell6:7-28. doi:10.1016/S1534-5807(03)00399-X
Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW. (1 Nov 2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature450, 56-62. doi:10.1038/nature06293
Antibiotics are used in a wide range of techniques in molecular biology. My aim with this post is to provide an easy reference to some of the main ones used in molecular biology, their mechanisms, range and working concentrations. I hope you will find it useful. Your Personal Antibiotics Reference Guide *Abbreviations: Gm(+/-)=Gram positive/negative; My=mycoplasma; […]
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