Topic: clones
+Anonymous A — 11.3 years ago #41,679
Clones of clones of clones
discus and pole vault
discus and pole vault
+Anonymous B — 11.3 years ago, 1 hour later[T] [B] #454,503
In 1962, biologist John Gurdon of Oxford University pioneered the method of the two step "nuclear transfer" process in frogs: the enucleation of a recipient oöcyte and the subsequent transfer of a differentiated somatic cell nucleus to that oöcyte. Gurdon’s experiments showed that despite the differentiated status of the donor nucleus, reconstituted cells appeared to reprogram, or dedifferentiate, the nucleus and enable it to function much as a naturally produced zygote. These zygotes successfully developed into viable embryos that hatched and grew into tadpoles. Because the tadpoles had all come from the gut cells of the same adult frog, they all had the same genetic material and thus were all clones. However, Gurdon’s nuclear transfer tadpoles clones failed to metamorphose into frogs. When scientists attempted to apply this technology to other species such as mice, cattle, or other mammals, the developmental program could not be reset (Gurdon and Uehlinger 1966; Byrne et al. 2002).
Scientists continued to tackle the problem and in 1986, Randall Prather and colleagues, then working in Neal First’s laboratory at the University of Wisconsin-Madison, cloned a cow from early embryonic cells using nuclear transfer (Prather et al. 1987). Although this was an example of blastomere nuclear transfer, it effectively set the stage for Dolly’s birth a decade later, on July 5, 1996. Dolly the sheep, the first organism ever to be cloned from adult cells, was created by Ian Wilmut and Keith Campbell using a technique similar to that used to create the first sheep from differentiated embryo cells (i.e., a blastomere clone) in 1995 (Wilmut et al. 1997).16
In July 1998, Ryuzo Yanagimachi, Toni Perry, and Teruhiko Wakayama of the University of Hawaii announced that they had cloned fifty mice from adult cells using the "Honolulu technique" (Wakayama et al. 1998). This was particularly significant because mouse embryos begin to divide almost immediately after the ovum is fertilized, and scientists had believed that this would not allow sufficient time for reprogramming to occur. Sheep, on the other hand, because their ova do not divide for several hours after fertilization, were thought to be an "easier" species to clone, as the natural delay between fertilization and division might be replicated in SCNT, possibly giving the oöcyte time to reprogram its new nucleus.
SCNT is a relatively new technology described by many as complex, technically demanding and inefficient, that continues to be developed and improved. As such, there is no set "method" that is universally employed, although the basic steps outlined below are common to most SCNT procedures at the time that this overview was written.
•Donor cell
For species in which the cloning process has been relatively well developed, the first step is identifying the animal to use as a nuclear donor. Animals to be used for breeding purposes are selected because they have been shown to be genetically superior to herd mates for the trait(s) to be propagated. Somatic cells can be collected from the ear (hole punch) or skin (surgical incision or needle aspiration), although many other cell sources have been used. Multiple factors may influence success or failure of the nuclear transfer process. Coordination of the cell-cycle stage of the donor nucleus and the recipient egg cytoplasm appears to be important for successful development of embryos. In general, the selection of a cell type for commercial cloning from an adult animal has evolved to choosing a collection method that is relatively noninvasive and minimizes stress to the live animal donor.
Several characteristics have been identified as contributors to the degree to which any given donor cell or type of cell will likely result in a successful cloning event (Sung et al. 2006; Oback and Wells 2007a). One example is the "replicative state" of the donor cell. In general, cells in culture accumulate nutrients, grow, and when they reach certain conditions, divide. Cells that adhere to a solid substrate, such as the bottom of a tissue culture dish tend to grow until there are so many of them that they begin to touch each other. Once that happens, they generally stop dividing, and go into a "resting state" with respect to replication (referred to as G0). Cells can also be directed into G0 by depleting the nutrients in their growth medium. Some laboratories have concluded that cells in G0 are the most effective donors (Wilmut and Campbell 1998, De Sousa et al. 2002). Conversely, other laboratories have found that actively dividing cells make good donors (Cibelli et al. 1998a, Lanza et al. 2001). Some laboratories find that cells from embryos or fetuses are the best donors (Batchelder 2005), while others are successful at cloning cells from aged or even deceased animals (Hill et al. 2000a, Tian et al. 2001). Another characteristic that has been shown to influence the degree to which cells make good donors is how "inbred" the donor animal is (Rideout et al. 2000). These researchers have determined that "hybrid vigor" is important for the success rate of animal cloning and the more inbred the donor animal, the less likely it is that cloning will occur successfully. Further, some species appear to be more amenable to cloning than others (e.g., goats compared with cattle, see Chapter V), and some species have not been cloned at all. At this time, the best conclusion that can be drawn with respect to the degree to which a cell (or animal) will serve as a "good" donor is that the technology is not sufficiently mature to predict with certainty which set of conditions will optimize cloning efficiency.
Once a cell has been isolated from culture, depending on the laboratory, either the entire cell or just its nucleus is transferred under the zona pellucida of the enucleated oöcyte using a very thin glass micropipette (Solter 2000) to await fusion.
•Oöcyte
The cell type used as the recipient for the donor cell to be cloned is the mature oöcyte, the version of the ovum that participates in fertilization during sexual reproduction. The oöcyte contains all of the non-nuclear cellular components required for the early development of an embryo. Oöcytes can be obtained from ovaries collected at slaughterhouses or from live animals using aspiration techniques (see previous discussion of in vitro fertilization). Because the oöcyte donates only its cytoplasm (the oöplast), it must be enucleated prior to fusion with the donor. The nucleus is generally removed by microaspiration, using a finely honed needle (PIFB 200317).
•Fusion
In order to begin the development process, the membranes separating the oöplast and the donor nucleus (or cell) must be fused. This can be accomplished in two ways: (1) by the administration of a brief electrical pulse, or (2) chemical fusion. Electrical stimulation appears to be the more commonly used technique and involves the application of one to several microbursts of a mild electrical current in the vicinity of the cells. This induces the formation of pores between the somatic donor cell and oöplast which functionally makes the two cells one. This process also stimulates embryonic development,18 which if successful, results in the development of blastocysts that are transplanted into surrogate mothers (Cervera et al. 2002).
Technical modifications aimed at increasing the success rate of cloning by improving the efficiency of the enucleation and fusion approaches are steadily evolving. For example, Oback et al. (2003) have developed a method that removes the zona pellucida from the oöcyte, aligns the donor cells with enucleated oöplasts, and uses electrofusion and chemicals to activate the cells to begin dividing. The results of this technique seem to show similar success rates for generating cattle clones as the cloning techniques more commonly used, with the advantage of being faster to perform (in the authors’ hands), and requiring less expensive equipment. Peura (2003) has also described a modified technique for preparing fused donor/oöplasts in which sheep oöcytes whose zona pellucidae had been removed were enucleated after fusion with donor cells, reversing the order in which those steps are usually performed. This technique appears to provide a higher rate of development of the blastocyst stage, implying that some factors present near the oöcyte chromosomes may be of assistance. Other factors that may influence the success rate of SCNT are the timing and method of embryo activation relative to fusion. For example, Sung et al. (2007) found that simultaneous electrical fusion and chemical activation of SCNT bovine embryos resulted in a higher rate of development to the blastocyst stage compared to embryos that were activated four hours postfusion. Compared to bovine SCNT embryos that were chemically activated, Schurmann et al. (2006) reported that postimplantation development of SCNT embryos was improved by using a non-chemical, more physiological method of activation (by transferring donor nuclei into enucleated oöcytes that had been fertilized in vitro).
Over the next few years other technical refinements may be developed, some based on improved technical practice, and others on increased knowledge about basic molecular mechanisms involved in the developmental process. These should increase the success rate of cloning, and decrease the potential for adverse events to occur.
•Transfer to recipient
Just as the case for other ARTs with an in vitro phase, the developing clone is transferred at the blastocyst stage into a surrogate dam in which the estrous cycle has been synchronized using standard methods. After transfer of the embryo clone(s), the pregnancy is allowed to proceed normally; no additional hormones or special treatments are required to establish or maintain pregnancy.
In cloning’s earliest days, the surrogate mother was often chosen to be distinctively different from the donor animal with respect to some clearly visible trait. For example, Dolly’s donor animal was a Finn Dorset sheep, a breed with white faces. Dolly’s surrogate mother, however, was chosen to be a black-faced sheep, so that if a white-faced sheep were born, it would be clear that it was not a genetic relative of the surrogate mother. In addition to choosing a distinctively different embryo recipient, Dolly’s identity was also confirmed by DNA fingerprint analysis of the donor cell line from which she was derived (Wilmut et al. 1997). DNA fingerprint analysis enables definitive confirmation that an animal clone was indeed derived from a specific cell type, and is now the method of choice for confirming genetic parentage of animal clones (First et al. 1994).
Scientists continued to tackle the problem and in 1986, Randall Prather and colleagues, then working in Neal First’s laboratory at the University of Wisconsin-Madison, cloned a cow from early embryonic cells using nuclear transfer (Prather et al. 1987). Although this was an example of blastomere nuclear transfer, it effectively set the stage for Dolly’s birth a decade later, on July 5, 1996. Dolly the sheep, the first organism ever to be cloned from adult cells, was created by Ian Wilmut and Keith Campbell using a technique similar to that used to create the first sheep from differentiated embryo cells (i.e., a blastomere clone) in 1995 (Wilmut et al. 1997).16
In July 1998, Ryuzo Yanagimachi, Toni Perry, and Teruhiko Wakayama of the University of Hawaii announced that they had cloned fifty mice from adult cells using the "Honolulu technique" (Wakayama et al. 1998). This was particularly significant because mouse embryos begin to divide almost immediately after the ovum is fertilized, and scientists had believed that this would not allow sufficient time for reprogramming to occur. Sheep, on the other hand, because their ova do not divide for several hours after fertilization, were thought to be an "easier" species to clone, as the natural delay between fertilization and division might be replicated in SCNT, possibly giving the oöcyte time to reprogram its new nucleus.
SCNT is a relatively new technology described by many as complex, technically demanding and inefficient, that continues to be developed and improved. As such, there is no set "method" that is universally employed, although the basic steps outlined below are common to most SCNT procedures at the time that this overview was written.
•Donor cell
For species in which the cloning process has been relatively well developed, the first step is identifying the animal to use as a nuclear donor. Animals to be used for breeding purposes are selected because they have been shown to be genetically superior to herd mates for the trait(s) to be propagated. Somatic cells can be collected from the ear (hole punch) or skin (surgical incision or needle aspiration), although many other cell sources have been used. Multiple factors may influence success or failure of the nuclear transfer process. Coordination of the cell-cycle stage of the donor nucleus and the recipient egg cytoplasm appears to be important for successful development of embryos. In general, the selection of a cell type for commercial cloning from an adult animal has evolved to choosing a collection method that is relatively noninvasive and minimizes stress to the live animal donor.
Several characteristics have been identified as contributors to the degree to which any given donor cell or type of cell will likely result in a successful cloning event (Sung et al. 2006; Oback and Wells 2007a). One example is the "replicative state" of the donor cell. In general, cells in culture accumulate nutrients, grow, and when they reach certain conditions, divide. Cells that adhere to a solid substrate, such as the bottom of a tissue culture dish tend to grow until there are so many of them that they begin to touch each other. Once that happens, they generally stop dividing, and go into a "resting state" with respect to replication (referred to as G0). Cells can also be directed into G0 by depleting the nutrients in their growth medium. Some laboratories have concluded that cells in G0 are the most effective donors (Wilmut and Campbell 1998, De Sousa et al. 2002). Conversely, other laboratories have found that actively dividing cells make good donors (Cibelli et al. 1998a, Lanza et al. 2001). Some laboratories find that cells from embryos or fetuses are the best donors (Batchelder 2005), while others are successful at cloning cells from aged or even deceased animals (Hill et al. 2000a, Tian et al. 2001). Another characteristic that has been shown to influence the degree to which cells make good donors is how "inbred" the donor animal is (Rideout et al. 2000). These researchers have determined that "hybrid vigor" is important for the success rate of animal cloning and the more inbred the donor animal, the less likely it is that cloning will occur successfully. Further, some species appear to be more amenable to cloning than others (e.g., goats compared with cattle, see Chapter V), and some species have not been cloned at all. At this time, the best conclusion that can be drawn with respect to the degree to which a cell (or animal) will serve as a "good" donor is that the technology is not sufficiently mature to predict with certainty which set of conditions will optimize cloning efficiency.
Once a cell has been isolated from culture, depending on the laboratory, either the entire cell or just its nucleus is transferred under the zona pellucida of the enucleated oöcyte using a very thin glass micropipette (Solter 2000) to await fusion.
•Oöcyte
The cell type used as the recipient for the donor cell to be cloned is the mature oöcyte, the version of the ovum that participates in fertilization during sexual reproduction. The oöcyte contains all of the non-nuclear cellular components required for the early development of an embryo. Oöcytes can be obtained from ovaries collected at slaughterhouses or from live animals using aspiration techniques (see previous discussion of in vitro fertilization). Because the oöcyte donates only its cytoplasm (the oöplast), it must be enucleated prior to fusion with the donor. The nucleus is generally removed by microaspiration, using a finely honed needle (PIFB 200317).
•Fusion
In order to begin the development process, the membranes separating the oöplast and the donor nucleus (or cell) must be fused. This can be accomplished in two ways: (1) by the administration of a brief electrical pulse, or (2) chemical fusion. Electrical stimulation appears to be the more commonly used technique and involves the application of one to several microbursts of a mild electrical current in the vicinity of the cells. This induces the formation of pores between the somatic donor cell and oöplast which functionally makes the two cells one. This process also stimulates embryonic development,18 which if successful, results in the development of blastocysts that are transplanted into surrogate mothers (Cervera et al. 2002).
Technical modifications aimed at increasing the success rate of cloning by improving the efficiency of the enucleation and fusion approaches are steadily evolving. For example, Oback et al. (2003) have developed a method that removes the zona pellucida from the oöcyte, aligns the donor cells with enucleated oöplasts, and uses electrofusion and chemicals to activate the cells to begin dividing. The results of this technique seem to show similar success rates for generating cattle clones as the cloning techniques more commonly used, with the advantage of being faster to perform (in the authors’ hands), and requiring less expensive equipment. Peura (2003) has also described a modified technique for preparing fused donor/oöplasts in which sheep oöcytes whose zona pellucidae had been removed were enucleated after fusion with donor cells, reversing the order in which those steps are usually performed. This technique appears to provide a higher rate of development of the blastocyst stage, implying that some factors present near the oöcyte chromosomes may be of assistance. Other factors that may influence the success rate of SCNT are the timing and method of embryo activation relative to fusion. For example, Sung et al. (2007) found that simultaneous electrical fusion and chemical activation of SCNT bovine embryos resulted in a higher rate of development to the blastocyst stage compared to embryos that were activated four hours postfusion. Compared to bovine SCNT embryos that were chemically activated, Schurmann et al. (2006) reported that postimplantation development of SCNT embryos was improved by using a non-chemical, more physiological method of activation (by transferring donor nuclei into enucleated oöcytes that had been fertilized in vitro).
Over the next few years other technical refinements may be developed, some based on improved technical practice, and others on increased knowledge about basic molecular mechanisms involved in the developmental process. These should increase the success rate of cloning, and decrease the potential for adverse events to occur.
•Transfer to recipient
Just as the case for other ARTs with an in vitro phase, the developing clone is transferred at the blastocyst stage into a surrogate dam in which the estrous cycle has been synchronized using standard methods. After transfer of the embryo clone(s), the pregnancy is allowed to proceed normally; no additional hormones or special treatments are required to establish or maintain pregnancy.
In cloning’s earliest days, the surrogate mother was often chosen to be distinctively different from the donor animal with respect to some clearly visible trait. For example, Dolly’s donor animal was a Finn Dorset sheep, a breed with white faces. Dolly’s surrogate mother, however, was chosen to be a black-faced sheep, so that if a white-faced sheep were born, it would be clear that it was not a genetic relative of the surrogate mother. In addition to choosing a distinctively different embryo recipient, Dolly’s identity was also confirmed by DNA fingerprint analysis of the donor cell line from which she was derived (Wilmut et al. 1997). DNA fingerprint analysis enables definitive confirmation that an animal clone was indeed derived from a specific cell type, and is now the method of choice for confirming genetic parentage of animal clones (First et al. 1994).
+Anonymous C — 11.3 years ago, 19 minutes later, 1 hour after the original post[T] [B] #454,505
+Anonymous D — 11.3 years ago, 1 hour later, 3 hours after the original post[T] [B] #454,515
dolly
+Syntax — 11.3 years ago, 3 hours later, 6 hours after the original post[T] [B] #454,525
+Syntax — 11.3 years ago, 1 minute later, 6 hours after the original post[T] [B] #454,526
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