Studies You Can Use

Kits for three of the gene-expression techniques published in 2006 are coming soon.

Jan 1, 2007
Jeffrey M. Perkel

Last January, I inaugurated this column with a look at three technology developments from 2005 that could soon be in kits. None are commercialized yet, but here are three more that should join them soon.

Let's start with a drug-inducible system for protein stabilization.1 Noting the lack of regulatory systems that act at the protein (as opposed to the nucleic acid) level, Stanford University researcher Tom Wandless, who says he likes to "invent new tools for biology," created his own.

Key to Wandless' system is a mutant FKBP12 protein that is degraded as soon as it's made, unless it is bound to a small-molecule ligand called Shield-1. "This new technique essentially allows investigators to use small molecules to regulate the expression levels of any protein of interest," Wandless writes in an E-mail. "We target proteins directly (not precursor DNA or RNA), so the method is considerably faster than transcriptional switches or RNAi. It is also dose-dependent, which means that the system is tunable with expression levels varying as a function of ligand concentration."

Wandless says his team has fused 26 different proteins to the so-called destabilization domain, "and we're 26 for 26" in inducing their degradation. Now he's adapting the system for live animal work. "We hope it will work in vivo and are currently testing it," he says. Wandless says Stanford's office of technology licensing is in negotiations with several companies to commercialize his system, though he adds he'll give it to anyone who asks.

Didier Trono and Patrick Aebischer of the Swiss Institute of Technology, Lausanne, also developed a drug-inducible gene-expression system. Whereas Wandless' method relies on protein stability, Trono and Aebischer's is based on chromatin modification.2 "It's basically a drug controllable system that acts through epigenetics to control any promoter," says Trono.

The system uses a lentiviral vector containing a series of tetracycline repressor-binding sites, a transgene, and a regulatory protein called TRKRAB, for fusion of the tetracycline repressor to a Kruppel-associated box domain. TRKRAB can recruit histone deacetylases and methylases to the DNA, converting it to heterochromatin over some two or three kilobases. The team built two variants, one that functions in the presence of tetracycline, and one that functions in its absence.

The system, Trono explains, can block transcription from polymerase II or III promoters, whether in cell culture, stem cells, or in vivo. It enables drug-controlled RNA interference. And, unlike standard tet-based regulatory systems, this one requires no promoter modification, "so it's far more flexible." Trono hasn't taken any steps to commercialize his technique. He has sent reagents to hundreds of colleagues; however, researchers can obtain them from Addgene (www.addgene.org), a Boston-based distribution firm, for $45 to $65 per plasmid.

Finally, from James Kadonaga, chair of molecular biology at the University of California, San Diego, comes a new promoter.3 "Core" promoters include the 80 or so nucleotides encompassing the RNA start site that direct the initiation of transcription by Pol II, and Kadonaga has spent about 15 years figuring out what makes them tick. Kadonaga says, "We thought, we have all these important motifs, what if we put them all into one core promoter? Would we get a super core promoter?" They did: the super core promoter SCP1 is the strongest ever made, five to 10 times more potent than the adenovirus major late-core or cytomegalovirus immediate early-core promoters in cells.

According to Kadonaga, SCP1 could prove useful anywhere amped-up protein expression is needed. The University of California has filed a patent application for the invention and now is looking to commercialize it. "There has been pretty good interest from some major companies," he says, but Kadonaga won't say which ones.

jperkel@the-scientist.com

References

1. L.A. Banaszynski et al., "A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules," Cell, 126:995-1004, Sept. 8, 2006. 2. J. Szulc et al., "A versatile tool for conditional gene expression and knockdown," Nat Methods, 3:109-16, February 2006. 3. T. Juven-Gershon et al., "Rational design of a super core promoter that enhances gene expression," Nat Methods, 3:917-22, November 2006.