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RESEARCH BRIEFSWhole Genes Delivered to CellsA gene without its native promoter, introns, and controlling elements is like a musical score without a time signature, rests and rhythm. Or maybe it is like a steak with the extra fat trimmed off. Who knows for sure? New Method for Highly Efficient Delivery of Large Human Genes
Researchers have developed a high-capacity infectious HSV-1 gene-delivery system that introduced a 115-kilobase human gene (plus a green fluorescent protein marker) to mouse liver cells in culture. The number of cells that took up the gene using the infectious system (far left) was much larger than the number that received the gene using three widely available chemical methods. Images courtesy of Richard Wade-Martins A new tool may help scientists study the nuances of a full gene, not just the cDNA excerpts transcribed into a working protein. To make a protein, a cell's enzymes typically edit out about 90 percent of the information along the length of a DNA strand that makes up a whole gene. In their labs, scientists usually follow suit. However, there may be something biologically important in those seemingly disposable chunks of non-protein-encoding regions. To investigate, researchers at Massachusetts General Hospital's Charlestown campus have developed the monster truck of gene delivery systems, which can deliver genomic DNA payloads as large as 150 kilobases into cells. In the November 2001 Nature Biotechnology, researchers in the lab of Antonio Chiocca, HMS associate professor of neurosurgery, show that large pieces of DNA, introns and all, were delivered intact and robustly expressed. "This is the first demonstration of efficient infectious delivery of a complete genomic locus over 100 kilobases, including all the regulatory elements within a gene as well as the coding DNA," said HMS instructor of neurosurgery Richard Wade-Martins, a Wellcome Trust International Travelling Research Fellow and first author on the paper. "We hope this will change the way people do gene expression experiments [for example, in functional genomics], because now we can deliver whole genes, not just the small coding regions, to a wide range of cell types for functional analysis." Earlier this year, senior author and HMS instructor of neurosurgery Yoshinaga Saeki reported in Molecular Therapy on the success of a refined virus-free packaging system using the oversized shell of the herpes simplex virus (HSV) as a gene shuttle. Meanwhile, Wade-Martins has been working with the bacterial artificial chromosomes--pieces of human DNA cloned in bacteria--from the human genome database. For this study, the researchers engineered a packaging signal into the entire 115-kilobase human HPRT gene, mutations of which can cause the neurodegenerative disorder Lesch-Nyhan syndrome. The viral vehicle loaded the gene in lieu of a virus. Then the system introduced the working gene into more HPRT-deficient human fibroblast cells and mouse liver cells than comparable chemical methods. "This work will provide a much needed tool for the analysis of gene complexity and may prove to be biologically useful in the discovery of drugs that affect gene function," Chiocca said. --Carol Cruzan Morton
Study Finds Genetic Link to Bone DensityAn international team of researchers has identified a gene that can affect bone strength. The discovery, reported in the Nov. 16 Cell, provides new insight into the growth and maturation of the skeleton and could one day lead to therapeutics for building bone in millions of people suffering from osteoporosis. "This is a very exciting and important discovery," said Bjorn Olsen, chair of the Forsyth Institute/Harvard School of Dental Medicine joint Department of Oral Biology and HMS professor of cell biology, in whose lab the research originated. The gene, LRP5, was isolated in studies of individuals affected by a rare but severe hereditary form of brittle bone disease, osteoporosis-pseudoglioma syndrome (OPPG). The disease begins in childhood, said principal researcher Matthew Warman, of the Howard Hughes Medical Institute and Case Western Reserve University School of Medicine. Located on chromosome 11, LRP5 encodes a receptor on the surface of bone-forming osteoblasts. Affected individuals have two nonworking copies of the gene. Their parents, who each carry a single mutant gene, appear predisposed to adult-onset osteoporosis, suggesting that the gene product could be an important therapeutic target for regulating bone mass. Blindness is another feature of the disease and further understanding of the role of LRP5 in the eye may provide new approaches for treating more common forms of vision loss. "The discovery provides important new information about how bone mass is regulated," said Olsen. "In addition, the gene is a very exciting candidate target for a set of new drugs to treat osteoporosis. Such drugs could actually reverse the effects of bone loss by stimulating the growth of bone-forming cells." --Anita Harris
Comprehensive Set of Photoreceptor Genes IdentifiedHMS researchers have identified nearly all the genes expressed in retinal cells, the key visual guides to the day- and night-time worlds. The discovery of the full set of photoreceptor genes, which was made in mice, could help establish an early warning system for the degenerative retinal diseases that rob the vision of millions of people worldwide. It could also lead to new methods for preserving and restoring the vision of those affected. Photoreceptors--including cones, which are activated by light, and rods, which operate only at night--are susceptible to a wide variety of inherited diseases, such as retinitis pigmentosa and cone-rod dystrophy. The newly identified gene set, reported in the Nov. 30 Cell, could lead to the identification of genes which, when mutated, cause these degenerative diseases. "We've cut down the search for disease genes by 100-fold," said Connie Cepko, HMS professor of genetics and senior author of the study. People at risk for inherited retinal disease could be screened for these mutants. The genetic database could lead to new methods for slowing or staying the course of a disorder once it occurs. "The more we know about how the genes work, the better we will be able to find ways to treat, and possibly, prevent disease," said Cepko. Studying how the multitude of genes works inside photoreceptors--which genes are turned on and when--could even lead to ways to replace dead or damaged cells. "Can we manipulate these genes in such a way to coax stem cells to become photoreceptors?" she asked. Though degenerative diseases of the retina had been mapped to 124 chromosomal regions, a little more than half of the actual mutant genes had been identified when Cepko, Seth Blackshaw, a postdoctoral fellow in Cepko's lab and lead author on the paper, and colleagues began their study. Using a computer program, SAGE, that compares snippets of genetic material taken from mouse retinal tissue with a huge genetic database from the mouse and human genomes, the researchers identified approximately 300 genes that were expressed specifically, or in an enriched manner, in photoreceptors--five times the number that were previously known. Two hundred sixty-four of these were newly identified. Of these, 241 had homologs in humans. "This makes photoreceptors the single most well-characterized cell type in the body," said Cepko. This information could be used to gain a better grasp not just of photoreceptors, which are highly specialized cells, but of other cells in the body. "We have very rich data regarding specialization," she said. "Do photoreceptors use specific proteins for various tasks? What kinds of processes in cells are being specialized?" While answers will take some time, retinal disease gene-hunters are likely to benefit from the genetic database immediately. "It is really a boon for human geneticists," said Cepko. "We had all these disease loci, and now we have a wonderful collection of candidate disease genes." --Misia Landau |
