BioWorld International Correspondent

LONDON Dutch researchers have invented a way of turning off single genes in cultures of mammalian cells and keeping them turned off.

The technology has been patented by The Netherlands Cancer Institute in Amsterdam, where the group works. They are holding discussions with biotechnology companies to set up a library of plasmids, each one designed to enter mammalian cells and turn off a specific gene, and they predict that this step should hasten scientists’ ability to systematically identify novel genes that are responsible for certain phenotypes.

Thijn Brummelkamp, René Bernards and Reuven Agami report their method in the March 21, 2002, online issue of Science in a paper titled “A system for stable expression of short interfering RNAs in Mammalian Cells.”

“Until now, it has not been possible to knock out genes in mammalian cells in an efficient way,” Agami, who is the group leader at The Netherlands Cancer Institute, told BioWorld International. “But this new method makes it possible to persistently inhibit gene expression for a long time, and so create loss-of-function phenotypes for biologists to study.”

The technology exploits and adapts a process that occurs normally during viral infections in lower eukaryotes. When a virus infects cells from these organisms, it produces long double-stranded RNA (dsRNA). The cell processes this dsRNA into shorter lengths, about 22 nucleotides long, called short interfering double-stranded RNAs (siRNAs). These shut down the activity of the genes being targeted by the virus, and prevent viral replication. Researchers realized that they could exploit this cellular machinery; adding long dsRNA for other genes also turned on the mechanism that shut down the genes concerned.

This method of inhibiting genes was good only for lower eukaryotes, however. If mammalian cells are infected with a virus that produces long dsRNA, the cells react with an interferon response. Ultimately, global protein production is shut down and the cells die.

Other groups had shown that introducing short dsRNA into mammalian cells could inhibit the gene from which the RNA was derived but the effect would last only for about four days.

“We wanted to overcome this transient effect and make the presence of siRNA stable in the cell, so that people could study loss-of-function phenotypes over long periods,” Agami said. The researchers set out to see whether they could induce the cells to manufacture the siRNA.

The trick was, Agami said, to take the required target sequence from a gene and clone it downstream of a promoter in such a way that, when transcribed into RNA, the oligonucleotide would form a stem-and-loop structure. This is achieved by ensuring that one end of the transcript naturally folds back and anneals with the other end of the transcript (thus forming the stem), leaving the loop in the middle. They ferried this construct into cells using either a normal plasmid, or a viral vector. Their name for these vectors is pSUPER, for suppression of endogenous RNA.

Experiments showed they could reliably turn off individual genes. For example, when they included siRNA against the p53 gene in the pSUPER and added this to mammalian cells in culture, they were able to show that the expression of the gene was significantly reduced, and that the cells behaved as though they had no p53. The loss of p53 persisted for the two months that the team continued testing.

pSUPER will, Agami predicts, be “quite a useful tool.” He and his colleagues also have shown that if there is even one point mutation in the gene sequence being targeted, the siRNA will not inhibit the gene. “In cancer, there are many genes where if there is a single point mutation, they become dominant and cause cancer. We will now be able to inhibit the expression of these genes specifically without affecting the normal allele,” Agami said. “It will then be possible to study what changes in these cells when, say, an oncogene is active and when it is inactive.” He also plans to inhibit tumor suppressor genes, to find out why cells that do not have functional copies of these genes become cancerous.

In addition, Agami said the technology may give rise to new therapies for infectious diseases. “Viruses such as HIV and influenza have their own genes and need some of the host’s own genes in order to proliferate,” he said. “The pSUPER method may make it possible to inhibit both viral genes of the virus and cellular genes that are vital for viral replication.” He acknowledged, however, that being able to deliver such therapies successfully will depend on overcoming the difficulties of delivering gene therapy in general.