Positive Reactions

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By Amber Lepage-Monette

PCR is an indispensable tool for two B.C.-based projects

For some researchers, polymerase chain reaction (PCR) is a commonplace part of their daily routines. For others, it can be a downright essential component in getting their projects completed.
Two B.C.-based initiatives are not only working with PCR, but using it to their advantage: the Mouse Atlas of Gene Expression Project and the Pleiades Promoter Project.
For the last four years, researchers from the Michael Smith Genome Sciences Centre (GSC) at the BC Cancer Agency (Vancouver, BC) have been working to develop a database, or “atlas” of gene expression profiles, officially wrapping up the Mouse Atlas of Gene Expression at the end of March.
“What the project involved was generating a database (that) had expression profiles of various tissues at various stages of development,” explains Pamela Hoodless, PhD. “We didn’t just want to profile the tissue in the adult, we also wanted to do gene expression profiling of the tissue as the embryo developed.”
Hoodless is part of the project’s mouse research team, a senior scientist at the Terry Fox Laboratory, and assistant professor of medical genetics at the University of British Columbia (UBC) (Vancouver, BC).
The Mouse Atlas will help researchers better understand gene expression, which in turn could improve understand of diseases that result from gene regulation failures.
The project’s goal was to build 200 Serial Analysis of Gene Expression (SAGE) libraries from tissues at various stages — from the fertilized egg to adult — and in different tissues, including heart, liver, pancreas and various brain regions.
SAGE libraries are collections of thousands of DNA “tags,” each representing a separate mRNA transcript. Sequencing the tags and identifying which genes they come from allows researchers to create a quantitative transcriptional profile of the tissue.
The SAGE libraries, as well as the software and analysis tools developed through the project, have been made available to researchers worldwide via the project’s website, and could prove beneficial to researchers in a variety of fields.
“You could take your genes of interest and just say, ‘where is my gene expressed?’ Or, if you’re interested in a specific pathway, you could look and say, ‘which components of the pathway are present in which tissues?’” Hoodless explains. “And you can do comparisons. If you wanted to look (at), for example, what genes are expressed exclusively in the kidney, you can take your kidney library and subtract all of the other libraries.”
For those working on the Mouse Atlas, the SAGE method allowed for more in-depth analysis, Hoodless says.
“Because SAGE is a method that is completely unbiased — in the sense that you don’t have to have a preconceived notion about what’s transcribed like you would with most spotted arrays — you can find transcription in areas (where) they weren’t necessarily thought to be transcribed before.”
PCR proved advantageous for the Mouse Atlas project because it allowed researchers to work with less tissue.
“It’s standard SAGE protocol (that) you need to start with 5 µg of total RNA,” Hoodless explains. “Now we’re down to about 200 ng of total RNA. And if we do a PCR amplification step . . . we can actually bring that down to 5 ng of total RNA.”
She goes on to explain that obtaining 5 µg of RNA requires approximately 5,000 fertilized eggs. By working with PCR techniques, the Mouse Atlas researchers were able to obtain the needed amount of RNA from approximately 200 eggs, greatly speeding up the process.
“It really allows us to look at organs and tissues where the amount that we can get is limiting,” she says.

The Next Step
While the Mouse Atlas project is now winding down, the Pleiades Promoter Project is just gearing up.
Launched this January, the project is funded over the next four years to build tools that will be used for gene therapy in several brain diseases, including Alzheimer’s, Huntington’s, depression, autism and addiction.
“We see gene therapy in all its various forms as a very important future direction for these kinds of diseases,” says Elizabeth Simpson, PhD, the Pleiades project leader, a senior scientist at UBC’s Centre for Molecular Medicine and Therapeutics, and an associate professor of medical genetics at UBC. Simpson is also a Canada Research Chair in Genetics and Behaviour.
“What we’ve tried to do is look at gene therapy as its being done today — in a very experimental form for the brain —and try to identify some of the aspects of the treatments that we think basic scientists could improve,” she says.
Three key areas the Pleiades project hopes to address are: the need for more clinical research into gene therapy; targeting genes to specific cell types to reduce side-effects; and conducting gene therapy in a controlled manner, inserting genes in specific locations of the genome.
As part of its research, the Pleiades team is looking specifically at the promoters that drive the genes being delivered in gene therapy.
Promoters are regions of DNA that control gene expression. There are two types of promoters: ubiquitous (which are expressed in every cell of your body) and specific (which are expressed in specific cells).
Simpson was also involved in the Mouse Atlas project. She created 67 region-specific brain SAGE libraries that the Pleiades team is using to find region-specific genes of the promoters in question.
Rob Holt, PhD, head of sequencing at the GSC and a senior scientist with the BC Cancer Agency, says PCR is integral to the project.
“We’re using PCR for all of the routine things, for amplifying genes and subcloning them into different vectors . . . so it’s a very important technique just for ordinary lab activities,” Holt says. “It’s hard to imagine how we’d get by without it.”
Simpson says that while PCR is used throughout the project, it is fusion PCR that is the enabling technology, without which the project would not be possible. Fusion PCR is a technique that brings together two pieces of DNA that are not found together naturally.
“You fuse or stitch them together to make what were originally non-contiguous pieces of DNA now contiguous,” Simpson explains. “It’s sort of an artificial promoter.”
With fusion PCR, the researchers capture a promoter’s motifs — which are responsible for the cell type of brain region — and fuse them together into one segment of DNA, and insert this minipromoter into a delivery vector for gene therapy.
“While the two motifs might be spaced out by 1,000 base pairs, or even . . . tens of thousands of base pairs in the genome, if you amplify them with primer tailed with (a) sequence that is complementary, you can then, in the next phase, do a secondary PCR reaction where both of those are combined into a single reaction. And the complimentary sequence at the ends of them actually primes the secondary reactions, so they are fused into a single molecule,” Holt explains.
This technique is much more streamlined and takes less time than traditional cloning methods, Holt says.
“It would take a couple of months to build one of these constructs using more traditional methods,” he says. “We can do it in about a week using this approach.”
The project is building four minipromoters for approximately 50 genes through fusion PCR.
“We’re developing tools that could be used for gene therapy,” Simpson says. “These will be synthetic human promoters that can be used for gene therapy research (in) these human diseases.”
The Pleiades project is seeking to put together a tool kit for gene therapy that will include the minipromoters, stem cell lines and software. Like the data coming out of the Mouse Atlas project, this tool kit will be made available to other researchers.
“One thing that we have committed to is that there will be no exclusivity in the distribution of these resources,” Simpson says. “They must be available to all basic researchers.”
For both the Mouse Atlas and Pleiades projects, PCR has proven to be an essential element in making this increased knowledge possible.
“It makes the whole, large-scale project much more tractable,” Holt says, particularly in reference to fusion PCR.
Simpson agrees. “Without it, this project couldn’t go forward,” she says.