E
Glossary

This glossary was developed by the panel from several sources, including the National Institutes of Health report, Stem Cells: Scientific Progress and Future Research Directions [1] and the National Bioethics Advisory Committee report Cloning Human Beings [2]. Boldface words in glossary definitions refer to other terms defined in the glossary.


Adult stem cell

– An undifferentiated cell found in a differentiated tissue in an adult organism that can renew itself and can (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated.

AI

– See Donor insemination

Amniocentesis

– A prenatal test performed by inserting a thin needle through the abdomen into the uterus and withdrawing a small amount of amniotic fluid (the fluid around the fetus) for laboratory testing. The fluid contains skin, kidney, and lung cells from the fetus that can be tested for chromosomal abnormalities, and the fluid itself can be tested for biochemical abnormalities. Amniocentesis is usually performed during the 15th week of pregnancy or later.

Andrology

– The science dealing with the structures, functions, and disorders of the male reproductive system.

Antigen

– Any substance or molecule that is recognized by the body as



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Scientific and Medical of Aspects: Human Reproductive Cloning E Glossary This glossary was developed by the panel from several sources, including the National Institutes of Health report, Stem Cells: Scientific Progress and Future Research Directions [1] and the National Bioethics Advisory Committee report Cloning Human Beings [2]. Boldface words in glossary definitions refer to other terms defined in the glossary. Adult stem cell – An undifferentiated cell found in a differentiated tissue in an adult organism that can renew itself and can (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated. AI – See Donor insemination Amniocentesis – A prenatal test performed by inserting a thin needle through the abdomen into the uterus and withdrawing a small amount of amniotic fluid (the fluid around the fetus) for laboratory testing. The fluid contains skin, kidney, and lung cells from the fetus that can be tested for chromosomal abnormalities, and the fluid itself can be tested for biochemical abnormalities. Amniocentesis is usually performed during the 15th week of pregnancy or later. Andrology – The science dealing with the structures, functions, and disorders of the male reproductive system. Antigen – Any substance or molecule that is recognized by the body as

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Scientific and Medical of Aspects: Human Reproductive Cloning “foreign” and that stimulates a specific immune response when it enters the tissues of an organism. ARTs – See Assisted reproductive technologies Artificial insemination – See Donor insemination Assisted reproductive technologies (ARTs) – Fertility treatments or procedures that involve laboratory handling of gametes (eggs and sperm) or embryos. Examples of ARTs include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Autoimmune disease or disorder – A category of diseases and disorders in which one’s own cells are mistakenly identified as “foreign” by the body and are therefore attacked by the immune system, causing tissue damage. Blastocoel – The fluid-filled cavity within the blastula. Blastocyst – A preimplantation embryo in placental mammals (about 3 days after fertilization in the mouse, about 5 days after fertilization in humans) of about 30–150 cells. The blastocyst stage follows the morula stage, and can be distinguished by its unique morphology. The blastocyst consists of a sphere made up of a layer of cells (the trophectoderm), a fluid-filled cavity (the blastocoel or blastocyst cavity), and a cluster of cells on the interior (the inner cell mass, or ICM). The ICM, consisting of undifferentiated cells, gives rise to what will become the fetus if the blastocyst is implanted in a uterus. These same ICM cells, if grown in culture, can give rise to embryonic stem cell lines. At the time of implantation the mouse blastocyst is made up of about 70 trophoblast cells and 30 ICM cells. Blastocyst cavity – The fluid-filled cavity within the blastocyst, sometimes referred to as the blastocoel. Blastomere – A cell from a morula-stage embryo. Blastula – Term (often used in lower vertebrates) to describe an early stage in the development of an embryo consisting of a hollow sphere of cells enclosing a fluid-filled cavity called the blastocoel. The term blastula sometimes is used interchangeably with blastocyst. Cell line – A general term applied to a defined population of cells that has been maintained in culture for an extended period and usually has undergone a spontaneous process, called transformation, that

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Scientific and Medical of Aspects: Human Reproductive Cloning allows the cells to continue dividing (replicating) in culture indefinitely. CGH – See Comparative genomic hybridization Chimera – An organism composed of cells derived from at least two genetically different individuals. Chorion – The outermost of the two membranes surrounding the embryo/fetus, part of which forms the fetal portion of the placenta. Chorionic villus sampling (CVS) – A prenatal test performed by removing a small sample of the placenta from the uterus with either a catheter (a thin flexible tube) or a needle. The sample can be tested for genetic abnormalities. Chorionic villus sampling is usually done between the 10th and 12th weeks of pregnancy. Chromosomes – Structures composed of very long DNA molecules (and associated proteins) that carry most of the hereditary information of an organism. Chromosomes are divided into functional units called genes, each of which contains the genetic code (instructions) for making a specific protein. A normal human body cell (somatic cell) contains 46 chromosomes; a normal human reproductive cell (gamete) contains 23 chromosomes. Cleavage – The process of cell division in the very early embryo before it becomes a blastocyst. Cleavage pattern – The pattern in which cells in a very early embryo divide; each species of organism displays a characteristic cleavage pattern that can be observed under a microscope. Departure from the characteristic pattern usually indicates that an embryo is abnormal, so cleavage pattern is used as a criterion for preimplantation screening of embryos. Clone – 1) An exact genetic replica of a DNA molecule, cell, tissue, organ, or entire plant or animal. 2) An organism that has the same nuclear genome as another organism. Cloning – The production of a clone. (For the purpose of this report, generating an individual animal or person that derives its nuclear genes from a diploid cell taken from an embryo, fetus, or born individual of the same species.) Comparative genomic hybridization (CGH) – A chromosomal screening technique that permits the detection of quantitative changes in chromosomal copy number without the need for cell culturing. It provides a global overview of chromosomal gains and losses

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Scientific and Medical of Aspects: Human Reproductive Cloning throughout the whole genome (including extra, missing, and broken chromosomes), but cannot detect small changes in DNA sequence or changes in the imprinting state of a gene. Culture – Growth of cells, tissues or embryos in vitro on an artificial nutrient medium in the laboratory. CVS – See Chorionic villus sampling Cytoplasm – The contents of a cell other than the nucleus. Cytoplasm consists of a fluid containing numerous structures, known as organelles, that carry out essential cell functions. DI – See Donor insemination Differentiated – Having developed into a specialized cell or tissue type Differentiation – The process whereby an unspecialized early embryonic cell or stem cell acquires the features of a specialized cell, such as a heart, liver, or muscle cell. Diploid – Refers to a cell having two sets of chromosomes (in humans, 46 chromosomes). In contrast, a haploid cell, such as a gamete, has only one set of chromosomes (23 in humans). DNA – A chemical, deoxyribonucleic acid, found primarily in the nucleus of cells (some is also found in the mitochondria). DNA is the genetic material that contains the instructions for making all the structures and materials the body needs to function. Chromosomes and their subunits, genes, are made (primarily) of DNA. DNA methylation – See Methylation Donor insemination (DI) or Artificial insemination (AI) – Deposition of sperm from a male donor inside a female reproductive tract for the purpose of achieving pregnancy. EBs – See Embryoid bodies EG cells – See Embryonic germ cells ES cells – See Embryonic stem cells Egg – The mature female reproductive cell. Embryo – A group of cells arising from the egg that has the potential to develop into a complete organism. In medical terms, embryo usually refers to the developing human from fertilization (the zygote stage) until the end of the eighth week of gestation when the beginnings of the major organ systems have been established.

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Scientific and Medical of Aspects: Human Reproductive Cloning Embryo splitting – Separation of an early-stage embryo into two or more embryos with identical genetic makeup, essentially creating identical twins or higher multiples (triplets, quadruplets, etc.). Embryoid bodies (EBs) – Irregularly shaped clumps of cellular structures that arise when embryonic stem cells or embryonic germ cells are cultured. Embryoid bodies usually contain tissue from all three of the germ layers: endoderm, mesoderm, and ectoderm. Embryoid bodies are not part of normal development and occur only in vitro. Embryonic germ (EG) cells – Pluripotent stem cell lines that migrate, during early development, to the future gonads to form the progenitors of egg or sperm cells. The properties of EG cells are similar to those of embryonic stem cells, but may differ in the DNA methylation of some imprinted regions. Embryonic stem (ES) cells – Primitive (undifferentiated) cultured cells from the embryo that have the potential to become a wide variety of specialized cell types (that is, are pluripotent). They are derived from the inner cell mass of the blastocyst. Embryonic stem cells are not embryos; by themselves, they cannot produce the necessary cell types, such as trophectoderm cells, in an organized fashion so as to give rise to a complete organism. Embryonic stem (ES) cell lines – Populations of dividing cells established from embryonic stem cells and cultured in the laboratory. Within embryonic cell lines are cells that can produce more embryonic stem cells or, under conditions of differentiation, give rise to collections of cells that include most or all cell types that can be found in a postimplantation embryo, fetus, or developed organism. Enucleation – A process whereby the nuclear material of a cell is removed, leaving only the cytoplasm. When applied to an egg, the removal of the maternal chromosomes, which are not surrounded by a nuclear membrane. Epigenetic effects – Changes in gene expression that occur without changing the DNA sequence of a gene; for example, in the epigenetic effect called genomic imprinting, chemical molecules called methyl groups attach to DNA and “turn off” the gene’s expression. Extraembryonic tissues – Intrauterine tissues derived from the zygote that support the embryo (for example, the placenta, the umbilical cord, and membranes such as the amniotic sac).

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Scientific and Medical of Aspects: Human Reproductive Cloning Fertilization – The process whereby male and female gametes (sperm and egg) unite. Fetus – 1) Legally, refers to the developing organism from the completion of implantation in the uterus to the time of birth. 2) In medical terms, refers to the developing human from the end of the eighth week to birth. At the end of the eighth week, the embryo is 2.0–3.0 cm (0.8–1.2 in.) long and weighs 1–4.5 g (0.04–0.16 oz). The major organ systems (for example, the nervous and cardiovascular systems) and rudiments of limbs, fingers, and toes have formed. Fibroblast – Cells that give rise to part of the connective tissue. Fluorescence in situ hybridization (FISH) – A technique that can be used for prenatal diagnosis, in which specifically designed fluorescent molecules are used to “light up” particular genes or sections of chromosomes to make them visible under a microscope. The fluorescence makes even small abnormalities in the chromosomes visible. Gamete – A reproductive cell (egg or sperm). Gametes are haploid (having only half the number of chromosomes found in somatic cells – 23 in humans), so that when two gametes unite at fertilization, the resulting one-cell embryo (zygote) has the full number of chromosomes (46 in humans). Gene – A functional unit of heredity that is a segment of DNA in a specific site on a chromosome. A gene directs the formation of a protein or RNA molecule. Gene expression – The process by which RNA and proteins are made from the instructions encoded in genes. Alterations in gene expression change the function of the cell, tissue, organ, or whole organism and sometimes result in observable characteristics associated with a particular gene. Genome – The complete genetic material of an organism. Genomic imprinting – See Imprinting Germ cell or Germline cell – A sperm or egg, or a cell that can develop into a sperm or egg; all other body cells are called somatic cells. Germinal vesicle transfer – See Oocyte nuclear transfer Germline cell – See Germ cell

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Scientific and Medical of Aspects: Human Reproductive Cloning Gestation – The period of development of an organism from fertilization of the egg until birth. Gonad – The reproductive organ that contains the developing sperm or eggs. The mature male gonads are the testes, and the mature female gonads are the ovaries. Graft-versus-host disease – A condition that occurs after tissue transplantation in which the donor-derived T cells attack the host’s tissues. Haploid – Refers to a cell (usually a gamete) having only one set of chromosomes (23 in humans). In contrast, body cells (somatic cells) are diploid, having two sets of chromosomes (46 in humans). Hematopoietic stem cell – A stem cell from which all red blood cells, white blood cells, and platelets develop. Heteroplasmy – See Mitochondrial heteroplasmy Identical twins – See Monozygotic twins Implantation – The process by which an embryo becomes attached to the inside of the uterus (7-14 days in humans). Imprinting – A process whereby DNA obtains biochemical marks that instruct a cell how and when to express certain genes. Imprinting often results in gene expression from only one copy of a gene – either the maternal or paternal copy. In utero – Latin: literally, “in the uterus.” In vitro – Latin: literally, “in glass”; in a laboratory dish or test tube; in an artificial environment. In vitro fertilization (IVF) – An assisted reproduction technique in which fertilization is accomplished outside the body. In vivo – Latin: literally, “in the living” subject; in a natural environment. Informed consent – A process in which a patient gives written consent (agreement) to undergo a medical procedure after having been provided with information about the nature of the procedure, risks, potential benefits, alternatives, and so on by his or her doctor. Inner cell mass – The cluster of cells inside the blastocyst. Before implantation, these can give rise to embryonic stem cell lines. After

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Scientific and Medical of Aspects: Human Reproductive Cloning implantation, the inner cell mass gives rise to all the tissues of the fetus, as well as some of the membranes around it. Institutional review board (IRB) – An administrative body in an institution (such as a hospital or university) established to protect the rights and welfare of human research subjects recruited to participate in research activities conducted under the auspices of that institution. The IRB has the authority to approve, require modifications in, or disapprove research activities in its jurisdiction, as specified by both federal regulations and local institutional policy. Intracytoplasmic sperm injection – An assisted reproductive method in which a sperm is injected directly into an unfertilized egg with a microscopic needle; this procedure is used in cases of severe male factor infertility. IVF – See In vitro fertilization Karyotype – The full set of chromosomes of a cell arranged with respect to size, shape, and number. This arrangement allows visual comparison of the chromosomes and identification of gross abnormalities (e.g. extra, missing or broken chromosomes). Major histocompatibility complex (MHC) – A group of genes that code for cell surface proteins that play a major role in histocompatibility (tissue compatibility; Latin: histo = tissue) in transplantation. Differences between the MHC proteins of a transplant donor and recipient are the major cause of transplant tissue rejection. Male factor infertility – Condition in which a male patient is infertile for such reasons as very low sperm count, sperm that cannot swim properly, sperm that are unable to penetrate the egg, or blocked sperm ducts. Meiosis – Cell division in the specialized tissues of ovaries and testes that results in the production of sperm or eggs, which contain half the number (23 in humans) of chromosomes found in somatic cells. During fertilization, the nuclei of the sperm and egg fuse to produce a zygote with the full number of chromosomes (46 in humans). Methylation – A biochemical process involving the addition of chemical tags called methyl groups (-CH3) to DNA. Methylation can be a signal for a gene or a section of a chromosome to turn off gene expression and become inactive or “silent.”

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Scientific and Medical of Aspects: Human Reproductive Cloning MHC – See Major histocompatibility complex Minor H antigens – See Minor histocompatibility antigens Minor histocompatibility antigens or Minor H antigens – A group of proteins (in addition to those encoded by the major histocompatibility complex (MHC) that can cause transplant tissue rejection. Minor H antigens can cause tissue rejection even when donor and recipient are matched for MHC. Immune response to minor H antigens is far less potent than response to MHC-encoded proteins, so the rejection is a slower process. Mitochondria – See Mitochondrion Mitochondrial heteroplasmy – An atypical condition characterized by the presence of more than one type of mitochondrial DNA in a single individual. Normally, each individual has only one type of mitochondrial DNA, inherited from his or her mother through the egg at fertilization. (Mitochondria from the sperm are systematically eliminated by the egg at fertilization.) Cloned organisms may exhibit mitochondrial heteroplasmy (having a mixture of mitochondria from both the donor cell and the recipient egg) because this elimination system may be bypassed during the cloning process. Mitochondrion (plural, Mitochondria) – A cellular structure in the cytoplasm that provides energy to the cell. Each cell contains many mitochondria. In humans, a single mitochondrion contains 37 genes on a circular mitochondrial DNA, compared with about 35,000 genes contained in the nuclear DNA. Monozygotic twins – Twins derived from one egg and one sperm (often called “identical twins”). Morula – The preimplantation embryo 3–4 days after fertilization, when it is a solid mass composed of 12–32 cells (blastomeres). After the eight-cell stage, the cells of the preimplantation embryo begin to adhere to each other more tightly, becoming “compacted.” The resulting embryo resembles a mulberry and is called a morula (Latin: morus = mulberry). Multipotent stem cells – Stem cells from the embryo, fetus, or adult, whose progeny are of multiple differentiated cell types and usually, but not necessarily, all of a particular tissue, organ, or physiological system.

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Scientific and Medical of Aspects: Human Reproductive Cloning Mutation – A change in DNA that alters a gene and thus the gene’s product, leading in some cases to deformity or disease. Mutations can occur spontaneously during cell division or can be triggered by environmental stresses, such as sunlight, radiation, and chemicals. Nuclear transfer – A procedure in which a nucleus from a donor cell is transferred into an enucleated egg or zygote (an egg or zygote from which the nucleus/pronuclei have been removed). The donor nucleus can come from a germ cell or a somatic cell. Nuclei – See Nucleus Nucleus (plural, Nuclei) – The compartment of a cell that contains the chromosomes. Oocytes – The developing female reproductive cells (the developing eggs) produced in the ovaries. Oocyte nuclear transfer or Germinal vesicle transfer – An assisted reproductive technique involving transfer of an egg nucleus (usually from a woman with age-related infertility or mitochondrial disease) into a healthy donor egg whose nucleus has been removed. This reconstituted egg can then be fertilized by a sperm in vitro. This technique may restore fertility to older women or to prevent the passing of mitochondrial disease to offspring. Ooplasmic transfer – An assisted reproduction technique that essentially enhances the defective (egg cytoplasm) from the patient’s egg with healthy cytoplasm from a donor egg. This “enhanced” egg can then be fertilized by a sperm in vitro. This procedure may restore fertility to older women. PCR – See Polymerase chain reaction PGD – See Preimpantation screening Placenta – A vascular organ-like structure that develops in the uterus during pregnancy, serving to anchor the embryo or fetus after implantation. The placenta enables oxygen and nutrients to pass from the maternal blood to the embryo or fetus. It also eliminates carbon dioxide and waste products from the embryo or fetus by passing them to the mother, who excretes them through her liver, kidneys, or lungs. Pluripotent stem cells (PSCs) – Stem cells that include in their progeny all cell types that can be found in a postimplantation embryo, fetus, or developed organism.

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Scientific and Medical of Aspects: Human Reproductive Cloning Polymerase chain reaction (PCR) – A technique for making multiple copies of a specific stretch of DNA or RNA; can be used to test for mutations in DNA. For example, if a stretch of DNA is mutated, the copies of it made with the PCR can be longer or shorter than normal. Precursor cells or Progenitor cells – In fetal or adult tissues, these are partially differentiated cells that divide and give rise to differentiated cells. Preimplantation embryo – The very early, free-floating embryo, from the time the egg is fertilized (zygote) until the beginning of implantation (in humans, a period of about 6 days). Also includes embryos resulting from nuclear transfer, in all the developmental stages through the blastocyst stage. Preimplantation screening or Preimplantation genetic diagnosis (PGD) – Before an in vitro-fertilized embryo is implanted in a woman’s uterus, it can be screened for specific genetic mutations that are known to cause particular genetic diseases or for chromosomal abnormalities. One or more cells are removed from the preimplantation embryo for testing. Prenatal diagnosis – Detection of abnormalities and disease conditions while a fetus is developing in the uterus. Many techniques for prenatal diagnosis, such as chorionic villus sampling and amniocentesis, require sampling placental tissue or fetal cells found in the amniotic fluid or fetomaternal circulation. Others, such as ultrasonography, can be performed without cell or tissue samples. Progenitor cells – See Precursor cells Pronuclei – See Pronucleus Pronucleus (plural, Pronuclei) – Refers to the haploid nucleus of egg or sperm prior to fertilization, and immediately after fertilization, before the sperm and egg nuclei have fused into a single diploid nucleus. Protein – A large complex molecule made up of one or more chains of amino acids. Proteins perform a wide variety of activities in the cell. PSC – See Pluripotent stem cells Recloning – See Serial nuclear transfer

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Scientific and Medical of Aspects: Human Reproductive Cloning Reprogramming – Resetting the developmental clock of a nucleus; for example, resetting the developmental state of an adult differentiated cell nucleus so that it can carry out the genetic program of an early embryonic cell nucleus, making all the proteins required for embryonic development. In somatic cell nuclear transfer, components of the recipient egg cytoplasm are thought to play an important role in reprogramming the somatic cell nucleus to carry out the functions of an embryonic nucleus. RNA (Ribonucleic acid) – A chemical that is similar in structure to DNA. One of its main functions is to translate the genetic code of DNA into structural proteins. Serial nuclear transfer or Recloning – The first step of this technique is a normal nuclear transfer, in which a nucleus is transferred into an enucleated egg, forming an embryo. In the second step, a nucleus from the resulting cloned embryo is transferred into another enucleated egg or an enucleated zygote (a fertilized egg with both male and female pronuclei removed). The second step can be repeated one or more times. This technique allows the nucleus to have two (or more) opportunities to be reprogrammed by egg cytoplasm (one during the original nuclear transfer, and more during subsequent nuclear transfers), thus potentially improving the chance of successful reprogramming of the nucleus. Somatic cell nuclear transfer (SCNT) – Transfer of the nucleus from a donor somatic cell to an unfertilized egg cell from which the maternal chromosomes have been removed. Somatic cell – Any cell of a plant or animal other than a reproductive cell or reproductive cell precursor. Latin: soma = body. Sperm – Mature male reproductive cells. Stem cells – Nonspecialized cells that have the capacity to divide indefinitely in culture and to differentiate into more mature cells with specialized functions. Stochastic – Random or involving a random variable. Telomerase – An enzyme composed of a catalytic protein component and an RNA template and that synthesizes the telomeric DNA at the ends of chromosomes. When active, telomerase can continually add to the length of the telomeres on the ends of chromosomes within a cell, thus conferring on that cell the ability to continue dividing past its normal lifespan.

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Scientific and Medical of Aspects: Human Reproductive Cloning Telomeres – “Caps” (made of repeated DNA sequences) found at the ends of chromosomes that protect the ends of the chromosomes from degradation. The telomeres on a chromosome shorten with each round of cell replication. Telomere shortening has been suggested to be a “clock” that regulates how many times an individual cell can divide (that is, when the telomeres of the chromosomes in a cell shorten past a particular point, the cell can no longer divide). Tissue culture – See Culture Totipotent cells – Stem cells that have unlimited developmental capability. The totipotent cells of the very early embryo (an embryo prior to the blastocyst stage) have the capacity to differentiate into extraembryonic tissues, membranes, the embryo, and all postembryonic tissues and organs. Transcription – Making an RNA copy from a gene or other DNA sequence. Transcription is the first step in gene expression. Transformation – A genetic process resulting in a heritable alteration of the properties of a cell. In the case of cultured cells, transformation often refers to the acquisition of new properties, such as unlimited culture lifespan. Translation – The process of forming a protein molecule from information contained in messenger RNA. Trophectoderm – The outer layer of the developing blastocyst that will ultimately form the embryonic side of the placenta. Trophoblast – The extraembryonic tissue arising from the outer layer of the blastocyst, involved in implantation and later in development of the placenta and chorion. Ultrasonography – Commonly called “ultrasound.” An imaging technique that uses high-frequency sound waves to create an image. During pregnancy, ultrasonography can be used to provide an image of the developing fetus, including the entire body, organs and surrounding tissue. Undifferentiated – Not having developed into a specialized cell or tissue type. Unipotent stem cell – A stem cell that both divides and gives rise to a single mature cell type, such as a spermatogenic stem cell, which only gives rise to sperm.

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Scientific and Medical of Aspects: Human Reproductive Cloning Uterus – The muscular pear-shaped organ (in humans, located in the lower part of a woman’s abdomen) in which the fetus develops. Vascular – Composed of or having to do with blood vessels. WGA – See Whole genome amplification Whole-genome amplification (WGA) – A technique that allows production of enough DNA from a single cell to do multiple genetic analyses; involves nonspecific polymerase chain reaction (PCR) amplification of an entire genome, providing templates for later PCR to produce more copies of the genome. X chromosome – One of the two sex chromosomes, the other being the Y chromosome. Females have two X chromosomes, and males have one X chromosome and one Y chromosome. X inactivation – Normal inactivation of one of the two X chromosomes in females. Y chromosome – The chromosome that determines male gender. Zygote – The one-cell embryo formed by the union of sperm and egg at fertilization. REFERENCES 1. US Department of Health and Human Services/ National Institutes of Health. Stem Cells: Scientific Progress and Future Research Directions. 2001. Online at: http://www.nih.gov/news/stemcell/scireport.htm. 2. National Bioethics Advisory Commission. Cloning Human Beings, Volume I: Report and Recommendations of the National Bioethics Advisory Commission. Rockville, MD. 1997. Online at: http://bioethics.gov/pubs/cloning1/cloning.pdf.