written by: Lamar Stonecypher•edited by: Leigh A. Zaykoski•updated: 4/3/2009
Variations on a theme: the different techniques used in animal cloning are more similar than they are different. However, they have become more efficient, and more common since Dolly. Did you know that Dolly was not the first cloned animal? That honor goes to her cousins Megan and Morag.
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Dolly was not the first cloned mammal. That honor goes to two of Dolly's cousins - Morag and Megan. Researchers at the Roslin Institute announced the birth of Morag and Megan in May, 1996. To clone the twins, they started with a nine-day-old embryo that had about one hundred cells. Early embryo cells have a property called totipotency, which is the ability to differentiate into any cell type, but these cells are still identical to their parent before they have differentiated. The research team, led by Ian Wilmut, allowed the totipotent embryo cells to divide in culture dishes, making thousand of cells in less than a week.
At the same time, hormone injections given to ewes made them produce many unfertilized eggs (secondary oocytes). The researchers surgically collected these oocytes and, in the laboratory, enucleated the oocytes (removed their cell nuclei).
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Nuclear transfer from the cultured totipotent cells into the enucleated oocytes was done by the process called electrofusion, which involves using a tiny electric pulse to fuse the cells together in culture. The fused cells then divided into a new embryo, using the genetic instructions from the totipotent cell line.
Two hundred and fifty embryos were implanted in Scottish blackface ewe surrogates. Of these, five survived to birth, three died within ten days, and only Morag and Megan survived past six months.
Megan and Morag were cloned from embryonic tissue, but Dolly was the first clone of an adult mammal. Dolly's birth was announced in February 1997, also at the Roslin Institute.
With Dolly, the researchers started with adult sheep mammary (breast) cells. These are different from embryonic cells in that they are already specialized. The differentiation that occurs to cell genes to make a nonspecialized cell into a specialized cell is through a process called DNA methylation. To remove the methylation, the Roslin team soaked the adult mammary gland cells in chemical baths that made them "forget" that they were specialized cells. Essentially, the cells were starved for about five days. During this time, the nuclei entered a "quiescent" stage, slowing the divisions of each nucleus's genetic material. Soon after, the nuclei were transferred into unfertilized egg cells through electrofusion, and these developed into embryos.
Out of 277 attempts, one of the embryos became Dolly.
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In 1998, Researchers at the Oregon Regional Primate Center cloned rhesus monkeys using the same electrofusion technique that produced Dolly, but cells from several different embryos were used, so the rhesus monkeys that were produced are siblings rather than twins. Notably, the experiment proved the nuclear transfer technology and duplicated Roslin's results. The animals, being primates, are also the most humanlike clones to date.
At the University of Wisconsin, a team led by Tanja Dominko cloned embryos from the skin cells of an adult cow (AgBiotech News & Information 1998). Taken from the cow's ear, the complete skin cells were inserted into enucleated cattle egg cells. An electric pulse was applied to fuse the two cells and make them start dividing into an embryo. These embryos were then implanted into cattle mothers.
In 1997, a company called ABSGlobal announced the birth of Gene, a Holstein bull calf, started from a thirty-day-old fetus (Infigen, Inc.). In their patented process, a cell was removed removed from the fetus and allowed to form a blastocyst of sixteen, thirty-two, or sixty-four cells. Then each of these cells was used to form a separate embryo .
As early as 1992, a company called Granada Biosciences was active in animal cloning from embryonic tissues. Their process entailed separating single cells from a growing calf embryo. Each cell was then injected into an enucleated egg cell and implanted in the womb of a surrogate cow. They experienced a twenty percent rate of success.
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In this, the second page of Remembering Dolly: Advances in Cloning Technology," we look at the Honolulu Method or micropipette method of cloning and a new method from Norway that has produced cloned piglets with human genes for Alzheimer's disease. honolulu method of cloning, cloning micropipette, norwegian microblade technique cloning
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In a process that is an improvement over the methods used with Dolly, Ryuzo Yanagimachi, of the University of Hawaii, has demonstrated a nuclear transfer technique that has come to be known as the Honolulu Method. This technique uses a micropipette (small glass tube) to insert the nuclear material. Working with mice, cloning is accomplished using nuclei from "cumulus cells," which surround a developing ovarian follicle. Using a micropipette, they inject donor nuclei from the cumulus cells into enucleated egg cells. Then, the eggs are prevented from dividing into blastocysts for five to six hours. This delay has proven crucial. Due to unknown cellular mechanisms, many more oocytes develop into blastocysts when the delay is introduced.
In a more recent development, seven piglets carrying human genes for Alzheimer ’s disease were born in Norway in 2007. Scientists Arne Lund Jørgensen at Aarhus University and Gábor Vajta at University of Copenhagen developed the specially modified piglets using a technique called “handmade cloning."
In this procedure, the donor egg cells outer shell, or zona pullicida is first dissolved with an enzyme. Then the genetic material – the chromosones and centrosome is identified, now on the surface of the cell, by direct observation using a microscope. A microblade is used to sever the chromosome and remove it, creating an egg without genetic material. Then, using a skin cell that carries the Alzheimer’s gene, an electrical shock is used to fuse the two cells together. After waiting one hour, the fused cells are merged with a second chromosome-free cell, and the egg then contains the complete genetic material from both the fibroblast and the skin cell. Electrical and chemical stimulation is provided to hasten cell division.Then the cells are cultivated in a special solution for zona pullicida free cells until they develop into a blastocyst.