Research Notes

More Efficient Cloning Scientists at the University of Connecticut have made a conceptual breakthrough in cloning by culturing nuclear donor cells for up to three months. Cloning had previously been successful only with fresh or short-term cultured cells. "It is shocking to us that we found long-term cultured cells not only can support development of offspring, but their efficiency for cloning is actually better than short-term culturing," comments Xiangzhong Yang, head of the University of Connecticut's transgenic animal facility. Yang and colleagues from Kagoshima Prefectural Cattle Breeding Development Institute, Japan, report in an upcoming issue of Proceedings of the National Academy of Sciences (PNAS) the generation of six genetically identical calves using skin fibroblast cells taken from the ear of a 17-year-old prize bull. This finding is pivotal to combining cloning technology with site-specific genetic manipulations, which are currently used to create new strains of mice with mutations in virtually any gene. Prolonged culture time may allow researchers to build a library of donor somatic cells with different genes removed for mammals other than mice. In this case, an altered nucleus may be transferred to an enucleated oocyte, which then would be implanted into a surrogate mother to create a transgenic animal. Researchers from the University of Hawaii, Honolulu, have also found cloning may have practical implications for manipulating the genome. Ryuzo Yanagimachi's lab has used extensively cultured embryonic stem (ES) cell lines to produce cloned mice (T. Wakayama et al., "Mice cloned from embryonic stem cells," PNAS, 96:14984-9, Dec. 21, 1999.) They demonstrated for the first time that the nucleus of a single ES cell could program full development, including placental formation.--Nadia S. Halim Lentivirus Limitation Researchers at Stanford University may have overturned an all but accepted dogma regarding the gene therapy applications of lentiviruses, a class of retroviruses that includes HIV. Gene therapy investigators have generally held that unlike other retroviruses, attenuated lentivirus vectors can target not only dividing cells, but also nondividing cells. But according to a recent paper, some degree of cell cycling in the liver is necessary for lentivirus efficacy, a property that could significantly limit the vector's usefulness in vivo (F. Park et al., "Efficient lentiviral transduction of the liver requires cell cycling in vivo," Nature Genetics, 24:49-52, January 2000). The Stanford team first suspected the vector's shortcoming when they had difficulties using lentivirus in the liver in a separate experiment. They subsequently found that the vector itself was the problem. According to senior author Mark Kay, an associate professor of pediatrics and genetics at Stanford, scientists will have to reconsider the way the lentivirus is currently being prepared and concentrated if they hope to improve in vivo applications. He suggests that something might copurify with the virus, or that the envelope used to package the vector may have some toxicity at high doses. Says Kay, "There may be ways to get around that, but I think that that's something that has to be addressed." His group is currently looking at lentivirus efficacy in other organs, trying to discover if the cell cycling requirement only applies to liver cells and T cells. --Eugene Russo Secretin Fails A single dose of synthetic human secretin is ineffective in treating autism or a similar condition called pervasive developmental disorder, the first full results to be published of a double-blind, controlled trial conclude (A.D. Sandler et al., "Lack of benefit of a single dose of synthetic human secretin in the treatment of autism and pervasive developmental disorder," New England Journal of Medicine, 341:1801-6, Dec. 9, 1999). Nevertheless, more than two-thirds of the parents of the study's 60 subjects told researchers they remain interested in the popular but unproven peptide hormone treatment for their children. Senior author James W. Bodfish, associate director of the Human Development Research and Training Institute at Western Carolina Center and associate professor of psychiatry at the University of North Carolina, points out that roughly one-third of the subjects responded, regardless of whether they received secretin or placebo. "The placebo phenomenon is robust in clinical science," he observes. "The mechanism of that, based on the way we conducted the study ... is largely the result of the expectations of the raters, who in this case were the parents." Standardized behavioral measures were used to rate changes in the children over four weeks. Bodfish says a secondary contributor to the placebo effect might have been responses of the children to behavioral changes toward them by their parents or teachers. The study did not find evidence that a subgroup within the responders was influenced by secretin rather than the placebo effect. Results of numerous other trials are yet to be published.--Steve Bunk Hunting X Escapees It's a chromosomal phenomenon in female mammals that's been known about for decades: During female embryonic development, one of the two X chromosomes in each embryonic cell condenses into a compact form called a Barr body and becomes inactive. Descendants of those embryonic cells therefore have either an active X chromosome derived from the mother or one derived from the father. But scientists have also known for some time that the phenomenon is not all-pervasive; some genes on the X chromosome somehow manage to escape X inactivation. As part of continuing efforts to find the mechanism of action of these "escapees," Case Western Reserve University researchers recently did the first systematic study of X inactivation status for a large number of X chromosome genes, an effort made possible by the increasing number of genes identified as part of the Human Genome Project (L. Carrel et al., "A first-generation X-inactivation profile of the human X chromosome," Proceedings of the National Academy of Sciences, 96:14440-4, Dec. 7, 1999). The team found that many more X-linked genes remain activated than previously thought: 34 of the 224 genes tested. Though many escapees on the X short arm had been identified previously, this was the first time researchers took a good look at the X long arm. Thirty percent of the short-arm genes studied appear to escape inactivation, compared to only 3 percent on the long arm. With this study and those previous, 10 percent of all X-linked genes have now been profiled. The other 90 percent remain to be done--and that's just from a high-throughput standpoint. According to lead author Laura Carrel, an instructor in the genetics department at Case Western, researchers will also dissect individual genes in an attempt to understand the roots of their expression.--Eugene Russo Knocking Down Genes Researchers at QIK Technology in Boston report the first successful use of antisense oligonucleotides (AS-ODNs) to inhibit in utero gene expression (S.E. Driver, "Oligonucleotide-based inhibition of embryonic gene expression," Nature Biotechnology, 17:1184-7, December 1999). This offers an alternative to traditional recombinant DNA knockout technology, which involves physically deleting a gene in an organism and studying the resulting phenotype. Unfortunately knocking out an essential gene may kill the organism before something can be learned about the gene function. "[AS-ODN] opens a whole new dimension in what we can do with mammalian models," comments Samuel Driver, president and chief operating officer of QIK Technology. With a synthetic DNA oligonucleotide that is complementary to the mRNA of a target gene, the researchers can suppress a gene at any point during the development of an organism. The oligonucleotide is chemically modified for in vivo stability and specific placental uptake. Driver and his colleagues studied the embryonic function of two essential murine genes, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and endothelial cadherin (E-cadherin). During development E-cadherin is first necessary for the implantation of the embryo. When the researchers waited to inhibit E-cadherin until after implantation, they saw neural tube defects, which are also lethal. Driver adds that this technology may be useful in modeling polygenic diseases, those that involve multiple defective genes. Several different AS-ODNs can be developed and used at the same time, so researchers can study the effect of knocking down multiple genes in a single animal.--Nadia S. Halim

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