BioWorld International Correspondent
LONDON - The ability to generate mammalian cells that have mutations in both copies of any particular gene could hasten exploration of the vast amount of information in the murine genome. One of the teams that discovered the method already has identified a new component of a DNA-repair pathway.
Allan Bradley, director of the Wellcome Trust Sanger Institute in Hinxton, UK, told BioWorld International: "Until we developed this approach, making mammalian cell lines that had both copies of a gene mutated was a very slow process."
The technique is described in a paper in the June 24, 2004, issue of Nature, titled "Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells." The method allows scientists to do in murine cells what they have been able to do in yeast cells and some whole animals for a while: knock out gene function. However, in mice, that involves creating mutant embryonic stem cells individually before breeding and cross-breeding the animals - a process that takes nearly a year.
With the new method, the first step is to create murine embryonic stem cells that lack the gene encoding Bloom's syndrome protein (Blm). Mutations in the gene are responsible for the human disease. Individuals with that disease tend to develop many breaks in their chromosomes.
Bradley's group previously had reported that Blm-deficient murine cells that have heterozygous mutations tend to give rise to daughter cells in which those mutations have become homozygous. For the work published in the Nature paper, Bradley, together with colleagues Ge Guo and Wei Wang, induced heterozygous mutations in the Blm-deficient cells using a retrovirus.
They affirmed that the mutations were caused by insertion of viral DNA by using a technique that allowed them to excise the viral DNA at will, and thus reverse the mutant phenotype.
Using that method, they generated a library of homozygous mutant cells that had mutations spanning the entire genome.
"We decided to look for mutants involved in DNA mismatch repair," Bradley said, "but researchers will be able to use this method to look for mutations in other types of pathways, which could be of interest from the point of view of basic research, or because they are clinically or commercially interesting."
The DNA mismatch-repair pathway, which is conserved from bacteria through to humans, has been well studied, but not all of the components involved in it have been described. To identify cells with mutations in those genes, the team made use of the observation that cells with a defect in the pathway would be resistant to a particular drug that normally causes mismatches.
"We started with about 10,000 different mutants, and we ended up with a handful that were in the DNA mismatch-repair pathway," Bradley said. "We found things we expected to find, such as the mismatch-recognition protein MSH6, which was known to be in this pathway, but we also found new genes not known to be in this pathway, including one called Dnmt1."
Further experiments showed that cells deficient in Dnmt1 have a feature called microsatellite instability, in which DNA-replication errors occur at a higher rate than normal. That finding confirmed for the researchers that the newly identified gene really does have a role in the mismatch-repair pathway.
Bradley and his group are planning additional screens. "For example," he said, "we believe we can identify genes which, when they are mutated, would prevent integration of a retrovirus into a host cell."
In the same issue of Nature, Junji Takeda and colleagues at Osaka University Medical School in Japan report on a related study. Their paper is titled "Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene."
The group used chemical mutagenesis with ENU, together with transient loss of expression of the Blm gene, to generate a similar library of embryonic stem cells, which had genome-wide mutations in both copies of each gene.
In their conclusion, the authors wrote: "Because pluripotent embryonic stem cells can differentiate into any type of tissue, a method for the comprehensive isolation of bi-allelic mutants should have a major impact on the analysis of molecular mechanism of differentiation in vitro as well as in vivo."