Pet cloning was based on the well-established method of nuclear transfer (see chapter 4). First, a blastomere was injected with nuclei from a mature oocyte. Then the injected cell was treated with ionomycin and cycloheximide to stop nuclear division. After about 14 hours, in which multiple mitotic cycles take place, the cell divides into two equal-sized daughter cells. The original blastomere had divided and developed a whole embryo without further nuclei. The two daughter cells were fused back into one cell and then developed into a clone, including both a blastocyst and an animal. These cell lines made headlines in 2008 when Dolly the sheep was cloned, using a method almost identical to that for producing Dolly (Scott, [@CR108]; Wilmut et al., [@CR136]). Chimeras (Blastomeres with Different Tissue Types) {#Sec4} ================================================== The first attempt to integrate a fully developed animal was made in 1966. A mouse embryo was divided into single cells by an enzyme (Dispase) and allowed to aggregate to a ball of cells. A piece of the ball was injected into the egg of a recipient female. A mouse hatched from this chimeric blastocyst (see chapter 4). Blastomeres without a cell wall were injected into an egg after removal of its zona pellucida by acid treatment. They then developed as blastocysts. These embryos were injected into mice and gave birth to healthy babies with a normal placenta. The development of the placenta and embryo were found to be identical to that of an embryo resulting from normal fertilization (Clark et al., [@CR32]). The creation of embryos from single-cell embryos by injection was achieved by Hirao in 2002 (Hirao et al., [@CR59]). With this method, a single cell from an eight-cell embryo can be injected to form a blastocyst and produce a viable animal with identical chromosomal constitution. These embryos were also injected into an empty mouse zona pellucida and implanted into a female mouse. No embryo develops without a uterus or uterine natural environment. Implantation is inhibited by the presence of another embryo in the uterus. Such chimera, in which two blastocysts develop, is called a heterotopic chimera, because the cells are derived from different embryos (hetero-) but develop at the same site (totoo). In another type of chimera, cells derived from a blastocyst and cells from another embryo are allowed to develop into separate animals in the same uterus (metachimerism). For example, if there is a one-year-old chimera that developed in a mouse's uterus from two embryos and chimeric blastocysts, this organism has a chimeric structure with separate organs derived from embryos of different ages. In a word, the two organisms are one inside the uterus. Chimeras of this kind can survive for years and even pass on to the next generation. Another type of chimera, the autochimer, arises from injecting an individual blastomere, not a group of blastomeres, into the enucleated egg (Hong et al., [@CR61]). This type of chimera can survive only briefly. This animal will be one that has lived inside the mother. Its cells are derived from an embryo that does not need the uterus for implantation. In 2006, a technique was introduced by Dr. Tseng's team in which a cloned blastocyst or blastomeres were embedded in a synthetic polymer that could be manipulated by chemicals in the medium. When treated with certain reagents, the surface of a clone was made into a layer of squamous epithelium, and then cells of different origins (embryonic or epithelial) were inserted (Li et al., [@CR76]). This chimera is called an isochorionic chimera. For chimera to develop properly, all the cells of the chimeric embryo must come from the same blastocyst (the original blastocyst that was separated). This is a strict rule because each clone contains cells with varying fates and behaviors. After fertilization, the egg becomes an independent individual, and the cells of the blastocyst can develop independently. For example, one cell can develop into an amniotic sac (with amniotic cells), another into an eye ball (with ocular cells), and so on. Because the tissue structures are different, some cells become trophoblast. Therefore, the developing blastocyst must be divided into small parts and put into three directions. When grown together, they will form an embryo and form an organ or an embryo itself. Barratt (1999) showed that when a normal blastocyst was divided into five to seven small parts, and the cells from these parts were returned into a larger blastocyst, there was a mosaic chimera. The cells of different ages grow independently at the same time. This chimera contains embryos of different ages. As the cells grow and develop, their growth behavior is different. The first chimera was observed by Hirao in 1986 when he separated cells from a blastocyst and transferred them into another mouse to observe what happened to them. He found that about 5--6% of the cells, either in a different location or in another organ of the same tissue (for example, in a different eye), grew and developed normally. However, this experiment could not be repeated more than twice, because the embryos would die. When the embryos were re-transferred into another uterus, the chimera could not develop and died. The first chimera, because it was a mosaic chimera, only had one possibility for growing: in one area it would become a normal embryo and then develop and grow to be a chimera. If in one area it became a chimera, the same was true for the other area. When they were transferred into a different uterus, they could develop normally. Chimera embryos from injected cells can not only be transferred back into the same mother, but they can also be transferred into another female mouse to create a new chimera. This process was first described by Koehler in 1993. In this case, an egg is removed from a recipient mouse with an empty uterus, and a number of isolated cells from a blastocyst are injected. Then, an embryo develops in the recipient's uterus, and the animal can later give birth to a healthy chimera. The success rate of the creation of chimeric clones is greater than that of cloning (90% vs. 80%). These clones are very interesting because they have different tissue types. The blood of the newborn chimera is the same as that of the mother who produced the cloned embryo. Chimeric animals have been known for over 30 years. However, their numbers are very small and are unlikely to have clinical relevance. As reported, in a cloned embryo, all the cells are of the same type. This embryo is called a meroblastic chimera. Because clones are obtained from a donor who has a cloned embryo, this clone must be also cloned. For example, the cloned cell-cloned embryos should have a complete genome of identical chromosomes. It is possible to clone one chimera using a single cell obtained from another chimera. A different type of chimera is called a parthenogenetic chimera, in which a fully developed embryo is cloned from a single cell of a single female cell clone, such as those from a human skin biopsy. The donor was cloned from this embryo, and parthenogenetic clones (which contain diploid female cells) were formed after many cell divisions. Parthenogenetic clones have the same number of chromosomes, and they can form normal clones. In 2002, the first animal clones were born using the technique of nuclear transfer. These clones are called parthenogenones. Using somatic cell nuclear transfer (see chapter 4), researchers were able to develop clones from a female animal. These cloned embryos were parthenogenones because they were the clone of a clone. They were parthenogenones because each egg contained a diploid number of chromosomes (2n). When two diploid cells are fused, they give rise to normal diploid cells and cannot develop into an embryo (Shen et al., [@CR113]). Clones with Nuclear Transfer Technology {#Sec5} ======================================= In 1996, Dolly the sheep was cloned (Wilmut et al., [@CR136]). Nuclear transfer (NT) is a process for producing artificial cells by fusing an egg cell with a mature somatic cell that has a complete genome. In normal embryonic development, the egg cell is fertilized by sperm to produce a totipotent egg cell, called a zygote. This cell has an equal number of maternal and paternal chromosomes. The zygote then goes through various cell divisions and gives rise to a complete organism. There are three stages of nuclear transfer (Nanoarchitecture and function of the sperm nuclear envelope, [@CR92]): (1) nuclear transfer, (2) embryonic development, and (3) birth of the cloned animals. In nuclear transfer, a somatic cell of the appropriate age with intact cell membranes is removed from the donor and injected into an enucleated oocyte. In NT, only one cell is used to produce an organism. As a result, it is easier to maintain this process than for the nuclear transfer to develop into multi-cell organisms. In addition to chromosomes, genes (DNA) from the donor are transferred into the nucleus of the enucleated oocyte (Ogilvie et al., [@CR97]). Next, the cloned embryo is cultured until the blastocyst stage. The embryo is then transplanted into the uterine horn of a surrogate mother with the assistance of an electric current to