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Elegant Knockdowns, DISC1, and Schizophrenia

Elegant Knockdowns, DISC1, and Schizophrenia

When I was a grad student—back in the Jurassic Era of molecular manipulations—my lab mates and I were all transfixed by the notion of a new technology: knockout animals (KOAs). This was because of its promise to solve a vexing problem. It was common in those days to isolate lots of genes, but determining their function was often a bewildering exercise. Not enough sequences had yet been placed in a database to make rational structure/function inferences using a computer. You could isolate a gene and then spend the rest of your life trying to figure out what it did.

That all began to change with KOAs. Using a variety of embryonic and molecular manipulations, it became possible to create genetically identical mice with typical populations with 1 critical exception: they were missing a single gene—your gene—if you knew how to make the animal. Your hypotheses about your gene’s function stood on much more solid ground with a simple inspection of the KOA’s anatomy, behavior, or both.

As useful as the technique became, it wasn’t a panacea; for people interested in developmental issues, knockout technologies always had a couple of annoying features. First was the inadvertent lethality problem. Many important genes play critical roles in the viability of the embryo, and if you knocked out these genes, the animal died. Not lots of utility there, particularly if you wished to look at postpartum behavior.

Second was the specificity problem. Gene products are usually expressed in many tissues. They even change functions depending on the developmental stage of the animal. Most investigators aren’t interested in the entire resum of a given protein, just the biological tasks currently being funded. When you create a KOA, you literally knock out the gene in every cell, not just in the tissue of interest. Not much specificity there. In the early days, knockout procedures could be performed with a very blunt instrument—a surprisingly risky investment. There was no guarantee you would get a living animal after the manipulation and no guarantee you could extract precise informa-tion from it if the creature somehow survived.

Things have changed radically in the ensuing years. More subtle molecular manipulations are available to people interested in, say, the distance between genes and behaviors. I would like to describe the fruit of one such technique. Describing the data won’t take much time, but we must review some basic molecular biology and a few techniques to understand what happened.

FOUR PIECES OF BACKGROUND INFORMATION

GFP

Short for green fluorescent protein, GFP is obtained from jellyfish; the protein encoded by this gene has a so-called reporter function. Its hallmark characteristic is an ability to turn any cell expressing it a bright green color. This “reporting” can be quite useful. GFP can be hooked up to the promoter sequence—an on/off switch—of a gene whose regulation is being studied, then placed inside a cell. If the cell turns green, the investigator knows the promoter was activated in that cellular background. The 3 researchers who discovered and characterized GFP (Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien) were awarded the 2008 Nobel Prize in Chemistry for their efforts.

Injection, followed by electroporation

It is one thing to construct a gene sequence using GFP; it is another to get that sequence solely into the nucleus of target cells of interest, and no others. The specificity problem mentioned with knockouts has been solved in a variety of ways, one of which is physical injection of the gene into the target region, followed by electroporation. Suppose you wanted to get GFP into the cells of a specific region of the brain of a lab animal, and in no other place. The first step is simply to inject the sequence of interest into the tissues you wish to target. This step does not put the gene sequence into the cells but simply places them near the desired location.

Next comes the electroporation step. Electroporation is a well-established technique whereby cells are exposed to an externally applied electrical field. Also called electropermeabilization, the field transiently opens up the cell plasma membrane, allowing foreign genes entrance into the cytosol. A certain percentage of cells that internalize the foreigner allows the genetic construct into the nucleus, where it becomes integrated into the host chromosome.

There are both desktop and handheld versions of these machines. In the handheld version, paddles mediating the electric field are supplied to either side of the target tissue. A current is then applied, opening up only those local cells. If the region has been previously injected with the gene construct of interest, say a GFP construct, some will find their way into the electroporated tissues. With the proper manipulation, they will begin to glow green. The gene of interest has been inserted into the tissue of interest, and in no other place. A version of this technology has been developed to allow for the insertion of genes into developing embryos. It is called in utero electroporation (IUEP).

RNAi and shRNA

RNAi stands for RNA interference. This technique exposes cells to a double-stranded RNA construct complementary to some target gene. This construct then triggers the degradation of the messenger RNA (mRNA) of the native target gene, thereby rendering it incapable of making protein. Indirect silencing occurs by the construct-stimulated activation of an interior nuclease (Figure). This protein degrades any mRNA sequence that is complementary to either strand of the introduced construct. It is a terrific way to transiently silence a specific gene. Like GFP, the elucidation of this technique led researchers Andrew Fire and Craig Mello to a Nobel Prize in 2006 (Physiology or Medicine).

A related construct and technology involves using an RNA construct called short hairpin RNA (shRNA). This, too, is engineered to be complementary to a gene of interest—and it, too, is double-stranded. Although synthesized as a linear sequence, its double-stranded nature occurs because of the construct’s tendency to auto-anneal, forming a tight, hairpin loop. This loop can be used to silence gene expression via the same interfering RNA system mentioned above.

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