A team of European researchers used nuclei from sheep somatic cells that had been killed by heat treatment to successfully clone four lambs. The research was published in the July 2002 issue of the journal Biology of Reproduction (http://www.biolreprod.org/). The authors announced that, to their knowledge, this is the first report of the cloning of mammals originating from nonviable cells. The team used nuclei from sheep granulosa cells that were fresh, fixed in methanol for thirty minutes, or heated in a 55 degree or 75 degree Celsius water bath for fifteen minutes. The nuclei were injected into egg cells which had their nuclei removed, harvested from anesthetized sheep. Cells that became embryos were transferred to a culture medium and cultures were maintained for eight days. At that time, embryos that had developed to the blastocyst stage were transferred into foster mothers who had been primed with hormones. Ninety bastocysts from the cloned embryos and 52 control embryos resulting from in vitro fertilization were transferred into recipient ewes. Twenty of the 26 ewes receiving the fertilized embryos became pregnant, and eighteen delivered offspring. Of the 43 ewes that received the cloned embryos, 31 were pregnant at day 40, but only six pregnancies remained viable until delivery. This success rate is similar to that of cloning using viable cells. Fetal loss was less frequent in the cloned embryos derived from heated cells, with four of the six cloned lambs born originating from nuclei treated with 55 degree heat, and one still surviving at the time of the writing of the article. The authors conclude that "cell viability is no longer an absolute requisite for cloning," and note "for the first time, that somatic cells rendered nonviable by heat are developmentally competent on nuclear transfer." This indicates that all that is necessary for a cell to be used in cloning is intact structure of the genetic material and a functioning centriole. The researchers state that the next step is to determine whether somatic cells that have been freeze dried can also be cloned. If this is possible, freeze-dried cells could be stored in normal refrigerators or possibly at room temperature, which would be helpful in establishing genetic banks from endangered species. This research could also be useful to scientists attempting to clone extinct animals.