Thursday, March 15, 2007

Baby, you're a knockout - RNA interference and Transgenic organisms

 One of the most powerful ways to find out what a gene does is to disable the gene in a seed, an ova or an embryo, grow the resulting 'transgenic' organism and find out what functions are missing.  This approach is being done for mustard seed and mice in order  to identify the function of each gene in these 'model organisms'.   Since many genes are re-used in other organisms, it is hoped that determining the function of each of the 29,500 mustard seed genes will shed insight into the genetics of other plants, and that determining the function of the murine genes in transgenic mice will translate into knowledge of the genetics of other mammals, like humans.  

1000s of varieties of mustard

From the NSF: To create a gene knockout, scientists use a bacterium called Agrobacterium to insert a code that tells a specific gene to turn off. According to Ecker, this process of T-DNA integration has been carried out for well over 25 years, but this study provides a new perspective on using the technique to analyze gene function.

Some genes, it turns out, contain certain features that mark them as favored targets of inactivation. Additionally, Ecker and his colleagues have discovered that fewer inactivations occur near the centromeres -- the thinner gene-poor regions of the chromosome. "These results provide significant new information in both the areas of functional genomics and basic plant biology," says Ecker. 

Of Mice and Men

There's an excellent article in The Scientist from 2002 that provides a history of the development of this technology, and this article provides details on the  painstaking procedures that were used to create transgenic mice.  But all that was before RNAi really took off. 

From another great article in The Scientist from 2003:   We've searched for decades for a way to knock out genes effectively," Sharp says. "We have about 35,000 different genes and have determined the function of only about 500 of them using an incredibly painful, expensive knockout technology. We won't ever understand the remaining functions with the existing technology."

From NeurologyReviews.com (Jan 2005):   Dr. Henry Paulson explained that RNAi has revolutionized investigation of gene activity in the lab. Simply by introducing the appropriately sequenced double-stranded RNA into a cell culture or animal model, it is possible to “knock down” production from any gene of interest.   “It’s now very clear that the most potent way to silence a gene is through a small double-stranded intermediate,” he said. The first demonstration of RNAi in mammals was in 2001, and since then, the field has exploded. In that seminal study, the nuclear envelope gene lamin was silenced. “Not only was it extremely potent but there were very few of the nonspecific effects seen with previous antisense technology,” he elaborated, referring to a related but entirely artificial technique. “The double-stranded RNA used like this does a heck of a lot better job.”

When RNA itself is directly introduced to the cell, however, it is eventually degraded. This may be useful in the lab for short-term studies, but for disease therapy, longer-term effects are desired. The alternative is delivering DNA that codes for the RNA, via a viral vector. “The advantage is that you have sustained expression within the cell of the double-stranded RNA you want,” said Dr. Paulson. “Can this work in the brain? Yes.”

2006 Nobel Prize in Medicine

The 2006 Nobel Prize in Medicine was awarded to Craig C. Mello and Andrew Z. Fire for their discovery of RNA interference - gene silencing by double-stranded RNA.  (illustrated presentation - check it out!)

From Craig C. Mello's HHMI web page

For decades, RNA molecules have been regarded as little more than DNA's messengers, ferrying the genetic code to the cell's protein-building factories. Craig Mello's research has helped to establish that certain RNA molecules play a far more impressive role in the cell. In a groundbreaking discovery, he found that short snippets of RNA can silence the expression of targeted genes. This phenomenon, called RNA interference, not only has become an indispensable means for studying gene function but has been found to be a normal part of gene regulation during embryonic development and may play a role in cancer and other diseases.

Craig was looking for an effective way of blocking the expression of specific genes in the developing embryo as a way to study their function. Working with C. elegans embryos, he injected RNA into the worms and was surprised to find that the interference effect was far more robust than expected. The RNA interference spread from cell to cell throughout the worm's body, regardless of the site of injection, and was transmitted from one generation to the next. "This was unheard of," Mello explained. "Something extremely interesting was going on but we didn't know what it was." After further studies conducted in collaboration with Andrew Fire of the Carnegie Institution of Washington, the pair revealed in a paper published in Nature in 1998 that the gene-silencing effect was in fact caused by double-stranded RNA.

From Stanford's web page 'The Secret Life of RNA':

The process of RNAi hinges on RNA’s Velcro-like nature. Like its sibling DNA, RNA is composed of a series of subunits called nucleotides designated A, U, G and C strung together in a chain. The series of letters making up each molecule determines what protein it generates and also allows it to clasp other RNA molecules or DNA. An A on one RNA molecule will find its match with a U on the other molecule and the C’s and G’s pair up. A molecule with the sequence ACUG, for example, would pair up with the opposing sequence UGAC.

 Although the fine details are slightly different in each organism, the broad brushstrokes of RNAi go like this: RNA molecules up to many thousands of letters long enter the cell where a protein called Dicer chops it into units of 21 or 22 letters. These chunks are called small interfering RNAs or siRNA. They conglomerate with proteins in the cell to make up the RNAi machinery. These complexes bind to the protein-producing RNAs within the cell that have a matching series of A’s, U’s, G’s and C’s. This binding marks the RNA molecule for destruction and eliminates the protein.

From the Novina Lab in Harvard:

In the triggering step of RNAi, Dicer cleaves long dsRNA into several siRNAs each with different sequences though all siRNAs are complementary to the triggering gene. In the effector step of RNAi, helicase activity in the RNA-induced Silencing Complex (RISC) unwinds the duplexed siRNAs and the antisense strand of the siRNA recruits RISC to target mRNA with exactly complementary sequence to the guide strand of the siRNA. An endonuclease in RISC degrades the targeted mRNA by cleavage of the mRNA at a position on the mRNA between the 10th and 11th nucleotides of the guide strand of the siRNA. The 3’ mRNA cleavage fragment possesses a 5’phosphomonoester.

 

From the Dana-Farber cancer institute: "Silence is golden" by Richard Saltus:

Unlike knockout mice, RNAi technology doesn't totally block genes' ability to make proteins — known as gene "expression" — but can reduce it to a very low level. Nevertheless, researchers use the terms gene "silencing" and "inactivation" when referring to RNAi.

"In simple terms, RNAi lets you inactivate any gene you're interested in at any time. This has rapidly become an essential and powerful tool," says Barrett Rollins, MD, PhD, Dana-Farber's chief scientific officer. "The specificity and flexibility of RNAi guarantee that it will become a standard and essential component of everyone's scientific toolbox."

In biology labs everywhere, RNAi tools are changing the way science is done. With a focus on cancer, scientists at Dana-Farber, along with colleagues at the Broad Institute of the Massachusetts Institute of Technology and Harvard University, have deployed RNAi technology in a hunt for genes they call cancer's "Achilles' heels" – genes behaving abnormally that tumors depend on to grow, survive, and progress. Using a specific type of RNAi tool called "short hairpin RNAs," or shRNAs, scientists can sequentially turn off genes in thousands of different cancer cells to find out which genes, when silenced, cause the cells to weaken or die. Identifying such genes would be the starting point for developing new drugs to target them.

Viral delivery of short hairpin RNAs:

Carl Novina, MD, PhD, used harmless viruses called lentiviruses to transport the RNA-interceptors into cells. "The advantage is that lentiviruses infect not just dividing cells, as many others do, but non-dividing cells where we want to use RNAi to find a gene's function," says Novina.  In addition, a gene silenced by an RNAi-carrying lentivirus stays silenced — a contrast to some other delivery techniques whose effects are transient. "It is now possible to use a lentivirus to infect an embryonic stem cell and generate a mouse in which the short hairpin RNA is expressed continuously," Novina says. "With the advent of specialized lentiviruses, it is possible to turn RNAi on and off at specific times to discover when genes function," he adds.

Video of RNA Interference (from the journal Nature)

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