Integrative Neuroscience Initiative on Alcoholism West, a National Institute of Alcohol Abuse and Alcoholism-funded consortium of 25 labs studying the genetic and neuroadaptive causes of alcohol dependence, has recently established a unit to integrate RNAi into the group’s effort.
According to Mike Zawada, an associate professor of clinical pharmacology at the University of Colorado Health Sciences Center and head of the consortium’s RNAi Core, the unit will use RNAi to knock down in vivo gene candidates identified over the past five years by INIA West labs in order to determine their role, if any, in alcoholism. (An initiative similar to INIA West called INIA East is ongoing, but the two operate independently of each other.)
“The main objective of the consortium is to understand the molecular basis for excessive alcohol consumption and craving for alcohol,” Zawada told RNAi News this week. “In the first five years, a large effort was placed on screening different strains of mice that, over decades, have been bred selectively to either prefer or dislike alcohol. These inbred stains were then subjected to gene array analyses for determining [the differences in gene expression between] the animals that prefer drinking versus those that dislike it.”
The result was a long list of genes that are differentially expressed between the so-called high alcohol-preferring and low alcohol-preferring mice, Zawada said. With the recent renewal of the consortium’s funding for another five years, “its goal is to keep discovering new potential differences in gene expression, but also attempting to confirm whether some of these changes really are meaningful in terms of the preference of the animals to drink.”
That is where RNAi comes into play, he said.
“The consortium is mostly interested in the [central nervous system] physiology — the brain control of drinking — so what we are proposing … is to utilize RNAi techniques to knock down selected genes [both] in specific brain areas and globally throughout the brain, and then to try to confirm whether these genes really play a role in the preference of the animals for alcohol,” Zawada explained.
However, with 24 separate labs (in addition to the RNAi Core) working on different aspects of alcoholism, ”there will be a lot of different genes and a lot of different ideas of what needs to be done first,” he noted. Through the RNAi Core, “we essentially have a unit within the consortium … [acting as] a unified depository of RNAi technologies that we’ll be using … to determine which genes have priority.”
Specifically, gene knockdown experiments proposed by consortium members will be evaluated by a project evaluation committee composed of members of INIA West’s steering committee, as well as an independent consultant. Also assisting the RNAi Core is Thermo Fisher Scientific subsidiary Dharmacon, which is providing reagents as well as its expertise in RNAi experiment design, Zawada added.
According to INIA West, the RNAi Core has three primary tasks: to perform high-throughput in vitro screening of RNAi sequences designed against the genes of interest; to silence expression of selected genes in mice and rats using RNAi in specific areas of the brain; and to examine the behavior and transcriptional effects of the silencing.
“The main objective of the consortium is to understand the molecular basis for excessive alcohol consumption and craving for alcohol.”
Although the RNAi Core will primarily use shRNAs expressed via lentiviral vectors and delivered to the brain through microinjections to do gene knockdown, Zawada said that siRNAs are also expected to come into play.
“The final objective is to be able to do a knockdown in the animals,” he said. “For that … we’re working with a cell type — neurons — that are generally post-mitotic and difficult to transfect in any way. Lentiviruses are one of the agents that are fairly successful in transduction of post-mitotic neurons,” so expressed shRNA will be the primary RNAi technology for the in vivo work.
However, with siRNAs “we can screen relatively quickly a number of candidate genes in the 96-well plate format that Dharmacon provides … for sequences that might be useful in creating the knockdowns in different neuronal cell lines and get a foot in the door, so to speak,” Zawada said. Additionally, there might be certain situations in which siRNAs delivered using an osmotic pump might prove more effective, depending on the nature of the experiments.
“Basically, we like to have the two platforms available” just in case, he added.
As for what projects the RNAi Core will tackle first, Zawada noted that the unit has just received its initial funding — $217,000 a year in direct costs — from the NIAAA.
“At this moment, we are just barely beginning,” he said.
INIA West Members
Richard Bell — Indiana University School of Medicine, Institute of Psychiatric Research
Susan Bergeson — University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research
Yuri Blednov — University of Texas Austin, Waggoner Center for Alcohol and Addiction Research
John Crabbe — VA Medical Center, Oregon Health & Science University
Christopher Cunningham — Oregon Health & Science University
Ron Davis — Baylor College of Medicine
Howard Edenberg — Indiana University School of Medicine
Nicholas Grahame — Indiana University School of Medicine
Adron Harris — University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research
Bob Hitzemann — Oregon Health Sciences University
Paula Hoffman — University of Colorado Health Sciences Center at Fitzsimons
Brigitte Kieffer — Institute of Genetics and Molecular and Cellular Biology (France)
George Koob — The Scripps Research Institute
Dayne Mayfield — University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research
Bill McBride — Indiana University Medical Center, Institute for Psychiatric Research
Rick Morrisett — University of Texas at Austin
Dolf Pfefferbaum — SRI International
Tamara Phillips — VA Medical Center, Oregon Health & Science University
Amanda Roberts — The Scripps Research Institute
Andrey Ryabinin — Oregon Health & Science University
George Siggins — The Scripps Research Institute
Karen Szumlinski — University of California, Santa Barbara
Boris Tabakoff — University of Colorado Health Sciences Center at Denver
Christine Wu — University of Colorado Health Sciences Center at Denver
Mike Zawada — University of Colorado Health Sciences Center at Denver