Automated approach to expression cloning

Functional genomics is exactly what it says on the tin: a growing field in which the staggering amounts of data gained from the multitude of international genomic sequencing projects are brought together to work out what it is that genes do.

Expression cloning is the one of the best methods available that allows scientists to ask some very important questions, to understand this core problem of what genes do.

Complementary DNA (cDNA) libraries using retroviral vectors have been used for this purpose for many years, but a consortium of researchers and institutes - the University of Queensland's Diamantina Institute, based at the Princess Alexandra Hospital campus in Brisbane, UQ's Institute for Molecular Bioscience and the University of Adelaide - have now come together to create a facility that uses this method in a much faster, simpler and more flexible way.

Tom Gonda and his colleagues call the project ARVEC, or arrayed retroviral expression cloning. The consortium has received several large grants from the NHMRC, the ARC and more recently from the Australian Cancer Research Foundation to establish a new, automated system at the Diamantina that they hope will allow Australian researchers to discover genes of biomedical importance.

"The idea of establishing this facility is to make it as generically applicable as possible," Gonda says. "It's not so much that we are designing it to do one specific type of assay or one kind of experiment. There are lots of people in the consortium and lots of our collaborators are interested in doing screens like this, so the idea is to make it as flexible as possible."

The project involves two stages: one, a robotics platform, is already up and running. The other is the creation of a cDNA library representing all of the genes in the human genome (although it will not be possible to represent all of the splice variants at this stage).

"The idea is to make a library, ultimately with all 20-odd thousand genes in the human genome represented, and to do this in an arrayed format, basically 96-well plates with one representative cDNA per gene," he says.

The consortium is not actually creating a new library, as it is far easier to buy a set of cloned open reading frames, which it has done.

"We've got about 12,000 at the moment, about half a genome's worth, and we'll hopefully acquire the rest as they are released. What we have to do now is to transfer each of those individually into a lentiviral vector and again we'll have a whole series of 96-well plates with one lentivirus containing one gene, and we will know where and which gene it is."

Robots will be used to transfect the viral vector DNAs, along with viral packaging constructs, individually into 96-well plates containing packaging cells. These will then make the lentivirus from the DNA, releasing the lentivirus into the supernate.

"It will incubate the cells - we have incubators as part of the robotics platform - and then we will harvest the virus from those cells, and as a separate run we will use that virus to transduce target cells, which after an appropriate incubation period will be assayed for whatever phenotype we are interested in."

The robotics platform includes a high-content imaging device, which works like an automated fluorescence microscope, as the readout system, he says.

"The kind of assays we use are those that have fluorescence as a readout in one way or another. Typically we will be staining cells with fluorescent antibodies. There are all kinds of things you can address doing that, but the main advantage of doing things this way is that we are not looking for cells that have any particular growth advantage or so on - we are only putting one gene in each well of target cells."

The consortium is currently fine-tuning the robotics platform, most of the components of which were provided by Millennium Science, and optimising processes such as virus production.

A small pilot library of about 1500 cDNA clones has been created and the first sets of transfections are now being tried.

"So far that's looking pretty good so the next step will be to screen this pilot library that we've made," Gonda says. "We are using some pretty simple screens just to show that it works."

A $3.2 million grant from the ACRF is helping further research, with part of the fund going towards new equipment for the new ACRF Comprehensive Cancer Genomics Facility, which will be based at the Diamantina and run in association with the Queensland University of Technology.

More robotics platforms are planned, as is the purchase of siRNA and shRNA libraries to identify drug targets.

---PB--- Investigating MYB

Besides organising robotics platforms, Gonda's real work is in his primary research interests in leukaemia and breast cancer.

He will use the screening technology to investigate genes that might affect differentiation of haemopoietic cells and the growth of breast cancer, for instance. Gonda has long studied the MYB gene, which was originally discovered in chicken leukaemia viruses and has some very good, and very bad, functions.

"MYB is essential for haemopoiesis ... but it is expressed in a large proportion of breast cancers, in leukaemia, and in colon cancers as well," he says.

"The interesting thing about those diseases and about where MYB is probably functioning in normal situations is that all of those three systems - blood development, developing mammary glands and colon - are rapidly turning over cell systems.

You are constantly making blood cells, constantly making gut cells and during pregnancy and lactation the mammary cells are proliferating rapidly and differentiating.

"So you have this coupled process of rapid growth and differentiation in all three of those systems. That's what we believe MYB is involved in - somehow this coupling of rapid growth and differentiation."

One area of interesting research involving MYB is that it is turned on by oestrogen. Last year, Gonda and colleagues showed how the oestrogen receptor turns on MYB and that MYB is actually necessary for the proliferation of oestrogen-receptor positive breast cancer cells.

The new robotics platform at the Diamantina will help further this research, he says. "One of the things that we can do with this system is that if you set up appropriate reporter assays you can look for genes that modulate the activity of a reporter.

"So for example, one of the things that we want to do is to look for genes that can affect either the activity or the control of expression of MYB. If we use a cell line that has a reporter for MYB we can put all of these genes in and can either over-express them or actually knock them out and see which one can affect MYB.

"One of things where not much is known about MYB, one of the big questions, is how MYB is actually regulated. People knew that MYB is turned off and on in certain circumstances but no one knew what any of the signals were and that's something of great interest. At least in the breast cancer cells we identified the oestrogen receptors as being what's upstream of MYB and we figured out how it actually worked.

"MYB's got an interesting mode of regulation - it turns out that instead of being regulated by the transcription actually starting, what happens is that the promoter of MYB is on all of the time in most cells but it is blocked in the middle of the first intron of the gene.

"So if MYB is switched on that block is overcome and that's what oestrogen and oestrogen receptors seem to be doing. We are interested in the signals that regulate MYB in maybe other cell types where it is turned on and off, say in blood cells or colon."