QUEBEC CITY, CANADA--Dolly the sheep made her entrance 8 years ago, but since then scientists have logged only spotty progress in creating others like her. Now, work presented here at the annual meeting of the Society for the Study of Reproduction, is providing new clues to what can go wrong in the earliest stages of cloning.
Despite all the excitement and apprehension that surrounds cloning, the process remains remarkably inefficient: Just 1% to 5% of cloned mammals survive. Those that are born alive often suffer an array of abnormalities, such as obesity and liver failure, and die young. Why so many perish is unclear, though researchers have found that many genes are either over- or underexpressed in cloned embryos (ScienceNow, 12 September 2002). Other recent work suggests problems in a key step of cell division: the formation and arrangement of spindles, stringy structures that divvy up the chromosomes for daughter cells.
To gain further clues, biochemist Keith Latham of Temple University in Philadelphia, Pennsylvania, and his colleagues focused on a mouse clone's earliest days, when the embryo is just one or two cells. To create cloned mice, the team inserted nuclei from so-called cumulus cells, which surround the ovary, into egg cells, or oocytes, without nuclei. Compared with normal embryos and unfertilized eggs that can be artificially stimulated to become embryos (a process called parthenogenesis), the clones exhibited some glaring defects.
For starters, a key protein called calmodulin, which helps spindles form, failed to bind to the spindles properly. Even when the oocyte's original spindles were left in, the defect remained--suggesting that lack of the original spindles may not be the only problem.
Another issue appears to be the broth in which the embryos are grown. Normally, clones are grown in culture used for embryos, and few clones flourish. Latham found that when the clones were immersed in another broth--one for oocytes and adult cells--roughly 50% of them survived.
Finally, a microarray analysis found that when a clone grew to two cells, 2700 messenger RNAs--indicators of various protein levels--were either over- or underexpressed. Many of those were so-called transcription factors, which can turn other genes on. Latham theorizes transcription factors "are the most difficult genes to reprogram" when creating a clone.
Latham's team has uncovered new clues about why cloning fails, says Alan Johnson, a physiologist at the University of Notre Dame in Indiana. The research could "enhance the efficiency" of cloning, he adds. Latham hopes so too. He believes that the defects he and his colleagues have found "can be overcome."