Tuesday, August 24, 2010

Human Genome Project

In 1990, the U.S. Human Genome Project begun formally. Department of Energy and the National Institutes of Health. The project originally was planned to last 15 years, but rapid technological advances accelerated the completion date to 2003. Project goals were to identify all the approximately 20,000-25,000 genes in human DNA, determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project.

To help achieve these goals, researchers also studied the genetic makeup of several nonhuman organisms. These include the common human gut bacterium Escherichia coli, the fruit fly, and the laboratory mouse.

A unique aspect of the U.S. Human Genome Project is that it was the first large scientific undertaking to address potential ELSI implications arising from project data.

Another important feature of the project was the federal government's long-standing dedication to the transfer of technology to the private sector. By licensing technologies to private companies and awarding grants for innovative research, the project catalyzed the multibillion-dollar U.S. biotechnology industry and fostered the development of new medical applications.

Landmark papers detailing sequence and analysis of the human genome were published in February 2001 and April 2003 issues of Nature and Science. See an index of these papers and learn more about the insights gained from them.

Discovery of Transposons

The chromosomal basis of heredity was already well established by the time McClintock began her graduate training in the Botany Department at Cornell University. Her experiments laid the groundwork for a serie of cytogenetic discoveries by the Cornell maize genetics group between 1929 and 1935. McClintock developed a method for using broken chromosomes to generate new mutations. Among the progeny of plants that had received a broken chromosome from each parent, she observed unstable mutations at an unexpectedly high frequency, as well as a unique mutation that defined a regular site of chromosome breakage. These observations so intrigued her that she began an intensive investigation of the chromosome-breaking locus. Within several years she had learned enough to reach the conclusion, published in 1948, that the chromosome-breaking locus did something unknown for any genetic locus: it moved from one chromosomal location to another, a phenomenon she called transposition. 
 
The study of transposable genetic elements and transposition became the central theme of her genetic experiments from the mid 1940s until the end of her active research career. This was incredulous at the time, DNA was believed to be stable and invariable. These jumping elements were isolated from the bacterium Escherichia coli in the late 1960's and were further defined as specific, small fragments of DNA which were given the name transposons. The scientific interest in transposons increased during the 1970's, when it appeared that they assisted in the transfer of bacterial resistance to antibiotics. Furthermore, it soon became evident that they caused most of the spontaneous mutations occurring in laboratory populations of more sophisticated organisms, such as yeast and the fruit fly. We now know that transposons are ubiquitous and may comprise up to 20% of an organism's genome.

Retrotransposons

Retrotransposons move by a "copy and paste" mechanism but in contrast to the transposons described above, the copy is made of RNA, not DNA.

The RNA copies are then transcribed back into DNA — using a reverse transcriptase — and these are inserted into new locations in the genome.

Many retrotransposons have long terminal repeats (LTRs) at their ends that may contain over 1000 base pairs in each.

Like DNA transposons, retrotransposons generate direct repeats at their new sites of insertion. In fact, it is the presence of these direct repeats that often is the clue that the intervening stretch of DNA arrived there by retrotransposition.

42% of the entire human genome consists of retrotransposons.

Transposons (Jumping Genes)

Grains of Indian corn come in different colors, such as purple, yellow and white. Sometimes the individual grains are purple with white streaks or mottling. This mottling effect defies Mendel's basic principles of genetics because individual grains may be multicolored rather than a single color. The movement of transposons on chromosomes may result in colored, non-colored and variegated grains that do not fit traditional Mendelian ratios based solely on chromosome assortment during meiosis and random combination of gametes. The explanation for this phenomenon involves "jumping genes" or transposons, and earned Dr. Barbara McClintock the prestigious Nobel Prize in Medicine in 1983 for her life-long research on corn genetics.

Transposons are genes that move from one location to another on a chromosome. In the pigmented aleurone layer of corn grains, the position of transposons may inhibit or block pigment production in some cells. For example, if the transposon moves to a position adjacent to a pigment-producing gene, the cells are unable to produce the purple pigment. This results in white streaks or mottling rather than a solid purple grain. The duration of a transposon in this "turned off" position affects the degree of mottling. If the pigmentation gene is turned off long enough by a transposon, the grain will be completely unpigmented. The reddish-purple patterns caused by transposons may be blotches, dots, irregular lines and streaks.
The following illustration shows how grain color in Indian corn may be affected by transposons. The different cards represent a linear sequence of genes on a chromosome. The ace of spades represents a transposon that moves to different positions on the chromosome. The jack of diamonds represents the gene for purple pigmentation in the corn grain. When the transposon (ace of spades) moves to a position adjacent to the gene for pigmentation (jack of diamonds), the pigmentation gene is blocked and no purple is synthesized (white area):

When the transposon (ace of spades) moves away from the gene for pigment production (jack of diamonds), the production of purple pigment is resumed (continuous purple area). In this example the gene for pigment production (jack of diamonds) is not adjacent to a transposon (ace of spades):

When a transposon moves to different positions within cells of the corn kernel, the coloration gene is "turned on" or "turned off" depending on whether it lands in a position adjacent to the pigmentation gene. Transposons may also have a profound effect on embryonic development and tumor formation in animal cells. Oncogenes (genes that cause tumors) may be activated by the random reshuffling of transposons to a position adjacent to the oncogene. Transposons may also be useful in genetic engineering with eukaryotic cells, by splicing in transposons to activate certain genes. The implications from Barbara McClintock's discovery of transposons may be far-reaching and as significant as Watson and Crick's discovery of the structure of DNA.

Human Gene Transfer

Human gene transfer is the process of transferring genetic material (DNA or RNA) into a person. DNA may be transferred as "naked" DNA, encapsulated DNA, or DNA within another organism, such as a virus. Use of retroviral vectors in humans also constitutes human gene transfer when the virus contains enzymes that result in a DNA copy of the RNA genome. 

Human gene transfer is experimental and is being studied to see whether it could treat certain health problems by compensating for defective genes, producing a potentially therapeutic substance, or triggering the immune system to fight disease. Human gene transfer may help improve genetic disorders, particularly those conditions that result from inborn errors in a single gene (for example, sickle cell anemia, hemophilia, and cystic fibrosis). It may also hold promise for diseases with more complex origins, like cancer and heart disease. Gene transfer is also being studied as a possible treatment for certain infectious diseases, such as AIDS. This type of experimentation is sometimes called "gene therapy" research. 

Scientists are attempting to determine whether human gene transfer can be safe and effective as a treatment for disease. Some experimental gene transfer procedures involve the introduction of DNA into cells, which then are injected into a person with disease. All such human gene transfer research studies require approval by the UCI Institutional Review Board (IRB), the UCI Institutional Biosafety Committee (IBC), and the NIH Recombinant DNA Advisory Committee (RAC).

Germline Gene Transfer

Gene transfer represents a relatively new possibility for the treatment of rare genetic disorders and common multifactorial diseases by changing the expression of a person's genes. Typically gene transfer involves using a vector such as a virus to deliver a therapeutic gene to the appropriate target cells. The technique, which is still in its infancy and is not yet available outside clinical trials, was originally envisaged as a treatment of monogenic disorders, but the majority of trials now involve the treatment of cancer, infectious diseases and vascular disease. Human gene transfer raises several important ethical issues, in particular the potential use of genetic therapies for genetic enhancement and the potential impact of germline gene transfer on future generations.
 
Scientific Issues
Gene transfer can be targeted to somatic (body) or germ (egg and sperm) cells. In somatic gene transfer the recipient's genome is changed, but the change is not passed on to the next generation. In germline gene transfer, the parents' egg and sperm cells are changed with the goal of passing on the changes to their offspring. Germline gene transfer is not being actively investigated, at least in larger animals and humans, although a great deal of discussion is being conducted about its value and desirability.
Many people falsely assume that germline gene transfer is already routine. For example, news reports of parents selecting a genetically tested egg for implantation or choosing the sex of their unborn child may lead the public to think that gene transfer is occurring, when actually, in these cases, genetic information is being used for selection, with no cells being altered or changed. In addition, in 2001 scientists confirmed the birth of 30 genetically altered children whose mothers had undergone a procedure called ooplasmic transfer. In this process, doctors injected some of the contents of a healthy donor egg into an egg from a woman with infertility problems. The result was an egg with two types of mitochondria, cellular structures that contain a minuscule amount of DNA and that provide energy for the cell. The children born following this procedure thus have three genetic parents, since they carry DNA from the donor as well as the mother and father. Although the researchers announced this as the "first case of human germline genetic modification," the gene transfer was an inadvertent side effect of the infertility procedure.

Gene Therapy

Gene therapy using an Adenovirus vector. A new gene is inserted into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.
Gene therapy may be used for treating, or even curing, genetic and acquired diseases like cancer
and AIDS by using normal genes to supplement or replace defective genes or to bolster a normal function such as immunity. It can be used to target somatic (i.e., body) or gametes (i.e., egg and sperm) cells. In somatic gene therapy, the genome of the recipient is changed, but this change is not passed along to the next generation. In contrast, in germline gene therapy, the egg and sperm cells of the parents are changed for the purpose of passing on the changes to their offspring.

There are basically two ways of implementing a gene therapy treatment:
1. Ex vivo, which means “outside the body” – Cells from the patient’s blood or bone marrow are removed and grown in the laboratory. They are then exposed to a virus carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the DNA of the cells. The cells are allowed to grow in the laboratory before being returned to the patient by injection into a vein.
2. In vivo, which means “inside the body” – No cells are removed from the patient’s body. Instead, vectors are used to deliver the desired gene to cells in the patient’s body.
As of June 2001, more than 500 clinical gene-therapy trials involving about 3,500 patients have been identified worldwide. Around 78% of these are in the United States, with Europe having 18%. These trials focus on various types of cancer, although other multigenic diseases are being studied as well. Recently, two children born with severe combined immunodeficiency disorder (“SCID”) were reported to have been cured after being given genetically engineered cells.
Gene therapy faces many obstacles before it can become a practical approach for treating disease.

DNA Sequencing,The Procedure

The DNA to be sequenced is prepared as a single strand.
This template DNA is supplied with
• a mixture of all four normal (deoxy) nucleotides in ample quantities
o dATP
o dGTP
o dCTP
o dTTP
• a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color:
o ddATP
o ddGTP
o ddCTP
o ddTTP
• DNA polymerase I
Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines). If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

Gene Expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA. The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea) and viruses - to generate the macromolecular machinery for life.

Several steps in the gene expression process may be modulated, including the transcription, RNA splicing, translation, and post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in a cell or in a multi cellular organism.
In genetics gene expression is the most fundamental level at which genotype gives rise to the phenotype. The genetic code is "interpreted" by gene expression, and the properties of the expression products give rise to the organism's phenotype.

DNA Profiling Techniques - Techniques For All Occasions

Most of us are familiar with the use of DNA profiling in criminal cases and with the establishment of the National DNA Database as an investigative tool on the back of this. DNA profiling can provide a high degree of confidence that a particular body fluid stain, hair or flake of dandruff, for instance, could have been deposited by one particular person. Indeed, a substantial part of Forensic Access’s work involves checking the accuracy and relevance of DNA test results and assessing the sustainability of conclusions drawn in respect of them as to likely culpability in specific case circumstances.
But the same sorts of techniques can have much wider applications, and these are discussed in this issue of Benchmark.

Establishing paternity
DNA analysis has been used in numerous paternity cases and is usually pretty straightforward. But one of the most challenging cases we have dealt with dates back to the 1950s, and has extended the frontiers of forensic DNA analysis.
The case concerned a man who, relatively late in life, discovered that the man that he had always thought was his father might not have been. Both his mother and his putative father were dead by the time his suspicions were aroused and he had no real clues to go on. One possibility for his paternity appeared to be a man living overseas with whom his mother had corresponded. Analysis of DNA in saliva on the backs of postage stamps on mail found amongst his mother’s possessions had the potential to hold the key – saliva that had been deposited more than 50 years ago!
Standard techniques produced weak, incomplete profiles of the DNA. It was not possible to exclude either of the men on the basis of these profiles, so we decided to attempt a new form of profiling that analyses DNA on the male-specific Y chromosome only. Y-chromosome profiles are passed on pretty much intact from father to son for generation after generation and so can provide a particularly powerful means of establishing paternity. In this case, the Y-chromosome profiling showed that the recent information was incorrect and indicated strongly that his father had been the man he always thought he was.

Identifying bodies or body parts
Over the years we have been increasingly successful in establishing the identity of human remains by applying our ever more sensitive DNA extraction and profiling techniques to bones, teeth and hair in particular; for example, one of HM Coroners wanted confirmation that a body, the remains of which had been found hanging in a wood, was who it was suspected to be.
Similar techniques can be used to resolve medical disputes; for instance, in a case where it was suspected that a mastectomy had been performed on the wrong woman. This was confirmed by comparing the DNA profile of a sample of the biopsy tissue taken from the woman in question with the profile of the excised breast tissue. Unfortunately, there had been a critical mix-up of samples in the hospital concerned.

Claims for compensation
We are often asked to help with claims for compensation made against manufacturers or retailers that relate to ‘contaminated’ products, especially foodstuffs. Very often the contamination involves some sort of body fluid such as blood on the wrappings or on the product itself. We regularly find ourselves examining yogurt pots, sandwiches or even, as on a recent occasion, an ice cream cone with the ice cream still inside it. In many cases, the contamination turns out to have come from the consumer – but not always. In these circumstances we may be asked to advise as to where in the supply chain the contamination could have occurred.
Contamination or sabotage of manufacturing production lines
Internally generated incidents, where disaffected or disillusioned employees relieve themselves on or into products, spit chewing gum onto them or even include sanitary tampons or used condoms in them, affect manufacturers in a very immediate way.
In one case we distinguished ourselves by obtaining a full DNA profile from urine that had been used in this way. This was a remarkable feat since urine does not normally yield much in the way of DNA-containing cellular material.
Very often the knowledge that the manufacturer has the means to identify which staff member is responsible is sufficient to persuade the guilty party to put their hands up and/or to leave, without the rest of the workforce having to have their DNA taken to be checked against the offending sample. 

Hate mail and general harassment
Because it is usually difficult to handle something without leaving some trace on it, we have achieved considerable success with analysing so-called hate mail and other items sent through the post as part of campaigns of general harassment. With mail, we tend to look first for saliva on the backs of postage stamps and envelope flaps, but the process does not end there. Each case is different and may present opportunities for evidence other than DNA to be found, which enables items to be ‘clustered’ as having come from a common source. In these cases, DNA profiling tends to form just one prong of the overall investigative strategy, albeit a very important one.

Other types of case
There are many types of circumstance where DNA profiling has been instrumental in resolving suspicions or matters in dispute. One of the more unusual ones concerned a medical doctor whose excessive prescription of certain drugs had been noticed. As part of the investigation, his consulting room was searched and injection needles were recovered from several safety disposal bins. Twenty needles were submitted for examination and blood in 10 of them was sent for DNA analysis. All 10 needles generated DNA profiles that matched the doctor’s own DNA profile, and it became clear that he had been taking the drugs himself.
The essential message is that DNA profiling has a much wider application range than might be appreciated at first sight, and it is always worth asking if it might be capable of providing answers, however old or unusual the circumstances surrounding the question.

DNA Banking

Our DNA banking service provides organizations and private individuals with the peace of mind that comes from knowing that their DNA samples are stored in a safe and highly secured environment. Banked DNA may be used for future DNA tests, for example:
• To protect against illegitimate claims on an individual’s estate
• To provide a standard for comparison and identification of people in high-risk professions, such as men and women in the military, law enforcement personnel, firefighters, and overseas contractors
• To assist with the identification of missing persons or give clues about the trail of a missing loved one
• To identify inherited traits, such as genetic diseases and other physical characteristics
Stored DNA provides a genetic history that will become vitally important as the genomic puzzle is completed. DNA from an elderly parent could one day provide clues about inherited diseases and other genetic issues. Some day very soon, this type of family tree knowledge could prove lifesaving.
Our services are completely confidential. We only release information on banked DNA to persons you authorize. There are two service options for you to choose from:
• Chain of Custody DNA Banking
• Chain of Custody DNA Banking and Profiling
Chain of Custody DNA Banking
In Chain of Custody DNA Banking, DNA samples are collected and stored using a process that ensures courts and other government agencies will consider the results of any future DNA testing on the stored DNA.
In compliance with Chain of Custody procedures, your DNA sample will be collected by a trained professional. Your DNA collection appointment will be scheduled at a hospital or laboratory near you. Upon banking your DNA, we will provide you with a banking certificate stating the storage period (15 years), the names of persons you authorize to retrieve or use your samples, and other important information.

Paternity Test


DNA paternity testing determines whether a man could be the biological father of a child. We all inherit our DNA (the genetic material) from our biological parents. A DNA paternity test compares a child’s DNA pattern with that of the alleged father to check for evidence of this inheritance—the most definitive proof of a biological relationship.

The result of a DNA paternity test is either an exclusion (the alleged father is not the biological father), or an inclusion (the alleged father is considered the biological father). For a standard paternity test, DDC guarantees at least 99.99% probability of paternity for inclusions or 100% certainty of exclusion.
Test Types: Legal and Home DNA Test

The type of paternity test you need will depend on what you intend to use the DNA test results for:

• If you need paternity test results that can be used as a legal document (for example, to change the name on the birth certificate or to obtain child support and other benefits), a Legal DNA Test needs to be performed (described below). 

• However, if you need the test only for personal knowledge, a Home DNA Test willl suffice.
Unlike the Home DNA Test, where tested parties collect their own samples at their convenience, the Legal DNA Test follows a Chain of Custody documentation process to ensure that you receive accurate and legally defensible results. When you set up your case with DDC, we will coordinate a convenient sample collection appointment, during which a trained sample collector will complete all the necessary documentation to satisfy chain of custody requirements.

DNA Testing: The DDC Advantage
DDC leads the DNA testing industry in its quality of service, which focuses on ensuring DNA test accuracy as well as a smooth experience for our clients. For all our DNA paternity testing clients, we:
• Run each test twice, following the most stringent procedures to guarantee accurate and conclusive results.
• Complete testing in 3 working days (5 working days for a prenatal test).
• Maintain confidentiality of each case using strict communication protocols.
• Schedule convenient appointments through our comprehensive network of collection sites.
DNA Test Participants
In a standard DNA paternity test, the tested parties include a child, the alleged father, and the mother (called a trio).
The mother’s participation in the paternity test helps to exclude half of the child’s DNA, leaving the other half for comparison with the alleged father’s DNA. However, we can perform a paternity test without mother’s participation (called a motherless). A motherless test involves additional analysis, which DDC performs without any additional charge. Results are equally conclusive whether or not the mother participates. Motherless tests are guaranteed to have at least a 99.9% probability of paternity for inclusions and 100% for exclusion.

Can Stem Cells Mend a Broken Heart?: Stem Cells for the Future Treatment of Heart Disease

Stem cells are a class of undifferentiated cells that are able to differentiate into specialized cell types. Commonly, stem cells come from two main sources:

Embryos formed during the blastocyst phase of embryological development (embryonic stem cells) and

Adult tissue (adult stem cells)

Heart disease which includes hypertension, coronary heart disease, stroke, and congestive heart failure, has ranked as the number one cause of death in the United States every year since 1900 except 1918. Nearly 2600 Americans die of CVD each day, roughly one person every 34 seconds.

The use of embryonic and adult-derived stem cells for cardiac repair is an active area of research. A number of stem cell types, including embryonic stem (ES) cells, cardiac stem cells that naturally reside within the heart, myoblasts (muscle stem cells), adult bone marrow-derived cells including mesenchymal cells (bone marrow-derived cells that give rise to tissues such as muscle, bone, tendons, ligaments, and adipose tissue), endothelial progenitor cells (cells that give rise to the endothelium, the interior lining of blood vessels), and umbilical cord blood cells, have been investigated as possible sources for regenerating damaged heart tissue. All have been explored in mouse or rat models, and some have been tested in larger animal models, such as pigs.

Tissue regeneration is probably the most important possible application of stem cell research. Currently, organs must be donated and transplanted, but the demand for organs far exceeds supply. Stem cells could potentially be used to grow a particular type of tissue or organ if directed to differentiate in a certain way. Stem cells that lie just beneath the skin, for example, have been used to engineer new skin tissue that can be grafted on to burn victims.

What are the potential uses of human stem cells and the obstacles that must be overcome before these potential uses will be realized?

There are many ways in which human stem cells can be used in research and the clinic. Studies of human embryonic stem cells will yield information about the complex events that occur during human development. A primary goal of this work is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs. Scientists know that turning genes on and off is central to this process. Some of the most serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation. A more complete understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. Predictably controlling cell proliferation and differentiation requires additional basic research on the molecular and genetic signals that regulate cell division and specialization. While recent developments with iPS cells suggest some of the specific factors that may be involved, techniques must be devised to introduce these factors safely into the cells and control the processes that are induced by these factors.
Human stem cells could also be used to test new drugs. For example, new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Other kinds of cell lines are already used in this way. Cancer cell lines, for example, are used to screen potential anti-tumor drugs. The availability of pluripotent stem cells would allow drug testing in a wider range of cell types. However, to screen drugs effectively, the conditions must be identical when comparing different drugs. Therefore, scientists will have to be able to precisely control the differentiation of stem cells into the specific cell type on which drugs will be tested. Current knowledge of the signals controlling differentiation falls short of being able to mimic these conditions precisely to generate pure populations of differentiated cells for each drug being tested.
Perhaps the most important potential application of human stem cells is the generation of cells and tissues that could be used for cell-based therapies. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.

What are induced pluripotent stem cells?

Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell–like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if iPSCs and embryonic stem cells differ in clinically significant ways. Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in late 2007. Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers.
Although additional research is needed, iPSCs are already useful tools for drug development and modeling of diseases, and scientists hope to use them in transplantation medicine. Viruses are currently used to introduce the reprogramming factors into adult cells, and this process must be carefully controlled and tested before the technique can lead to useful treatments for humans. In animal studies, the virus used to introduce the stem cell factors sometimes causes cancers. Researchers are currently investigating non-viral delivery strategies. In any case, this breakthrough discovery has created a powerful new way to "de-differentiate" cells whose developmental fates had been previously assumed to be determined. In addition, tissues derived from iPSCs will be a nearly identical match to the cell donor and thus probably avoid rejection by the immune system. The iPSC strategy creates pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

What are the similarities and differences between embryonic and adult stem cells?

Human embryonic and adult stem cells each have advantages and disadvantages regarding potential use for cell-based regenerative therapies. One major difference between adult and embryonic stem cells is their different abilities in the number and type of differentiated cell types they can become. Embryonic stem cells can become all cell types of the body because they are pluripotent. Adult stem cells are thought to be limited to differentiating into different cell types of their tissue of origin.
Embryonic stem cells can be grown relatively easily in culture. Adult stem cells are rare in mature tissues, so isolating these cells from an adult tissue is challenging, and methods to expand their numbers in cell culture have not yet been worked out. This is an important distinction, as large numbers of cells are needed for stem cell replacement therapies.
Scientists believe that tissues derived from embryonic and adult stem cells may differ in the likelihood of being rejected after transplantation. We don't yet know whether tissues derived from embryonic stem cells would cause transplant rejection, since the first phase 1 clinical trial testing the safety of cells derived from hESCS has only recently been approved by the United States Food and Drug Administration (FDA).
Adult stem cells, and tissues derived from them, are currently believed less likely to initiate rejection after transplantation. This is because a patient's own cells could be expanded in culture, coaxed into assuming a specific cell type (differentiation), and then reintroduced into the patient. The use of adult stem cells and tissues derived from the patient's own adult stem cells would mean that the cells are less likely to be rejected by the immune system. This represents a significant advantage, as immune rejection can be circumvented only by continuous administration of immunosuppressive drugs, and the drugs themselves may cause deleterious side effects

What are adult stem cells?

An adult stem cell is thought to be an undifferentiated cell, found among differentiated cells in a tissue or organ that can renew itself and can differentiate to yield some or all of the major specialized cell types of the tissue or organ. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found. Scientists also use the term somatic stem cell instead of adult stem cell, where somatic refers to cells of the body (not the germ cells, sperm or eggs). Unlike embryonic stem cells, which are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in some mature tissues is still under investigation.
Research on adult stem cells has generated a great deal of excitement. Scientists have found adult stem cells in many more tissues than they once thought possible. This finding has led researchers and clinicians to ask whether adult stem cells could be used for transplants. In fact, adult hematopoietic, or blood-forming, stem cells from bone marrow have been used in transplants for 40 years. Scientists now have evidence that stem cells exist in the brain and the heart. If the differentiation of adult stem cells can be controlled in the laboratory, these cells may become the basis of transplantation-based therapies.
The history of research on adult stem cells began about 50 years ago. In the 1950s, researchers discovered that the bone marrow contains at least two kinds of stem cells. One population, called hematopoietic stem cells, forms all the types of blood cells in the body. A second population, called bone marrow stromal stem cells (also called mesenchymal stem cells, or skeletal stem cells by some), were discovered a few years later. These non-hematopoietic stem cells make up a small proportion of the stromal cell population in the bone marrow, and can generate bone, cartilage, fat, cells that support the formation of blood, and fibrous connective tissue.
In the 1960s, scientists who were studying rats discovered two regions of the brain that contained dividing cells that ultimately become nerve cells. Despite these reports, most scientists believed that the adult brain could not generate new nerve cells. It was not until the 1990s that scientists agreed that the adult brain does contain stem cells that are able to generate the brain's three major cell types—astrocytes and oligodendrocytes, which are non-neuronal cells, and neurons, or nerve cells.
A. Where are adult stem cells found, and what do they normally do?
Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis. They are thought to reside in a specific area of each tissue (called a "stem cell niche"). In many tissues, current evidence suggests that some types of stem cells are pericytes, cells that compose the outermost layer of small blood vessels. Stem cells may remain quiescent (non-dividing) for long periods of time until they are activated by a normal need for more cells to maintain tissues, or by disease or tissue injury.
Typically, there is a very small number of stem cells in each tissue, and once removed from the body, their capacity to divide is limited, making generation of large quantities of stem cells difficult. Scientists in many laboratories are trying to find better ways to grow large quantities of adult stem cells in cell culture and to manipulate them to generate specific cell types so they can be used to treat injury or disease. Some examples of potential treatments include regenerating bone using cells derived from bone marrow stroma, developing insulin-producing cells for type 1 diabetes, and repairing damaged heart muscle following a heart attack with cardiac muscle cells.
B. What tests are used for identifying adult stem cells?
Scientists often use one or more of the following methods to identify adult stem cells: (1) label the cells in a living tissue with molecular markers and then determine the specialized cell types they generate; (2) remove the cells from a living animal, label them in cell culture, and transplant them back into another animal to determine whether the cells replace (or "repopulate") their tissue of origin.
Importantly, it must be demonstrated that a single adult stem cell can generate a line of genetically identical cells that then gives rise to all the appropriate differentiated cell types of the tissue. To confirm experimentally that a putative adult stem cell is indeed a stem cell, scientists tend to show either that the cell can give rise to these genetically identical cells in culture, and/or that a purified population of these candidate stem cells can repopulate or reform the tissue after transplant into an animal.

What are embryonic stem cells?

C. What laboratory tests are used to identify embryonic stem cells?

At various points during the process of generating embryonic stem cell lines, scientists test the cells to see whether they exhibit the fundamental properties that make them embryonic stem cells. This process is called characterization.

Scientists who study human embryonic stem cells have not yet agreed on a standard battery of tests that measure the cells' fundamental properties. However, laboratories that grow human embryonic stem cell lines use several kinds of tests, including:

* Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term growth and self-renewal. Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated.
* Using specific techniques to determine the presence of transcription factors that are typically produced by undifferentiated cells. Two of the most important transcription factors are Nanog and Oct4. Transcription factors help turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development. In this case, both Oct 4 and Nanog are associated with maintaining the stem cells in an undifferentiated state, capable of self-renewal.
* Using specific techniques to determine the presence of paricular cell surface markers that are typically produced by undifferentiated cells.
* Examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed. It does not detect genetic mutations in the cells.
* Determining whether the cells can be re-grown, or subcultured, after freezing, thawing, and re-plating.
* Testing whether the human embryonic stem cells are pluripotent by 1) allowing the cells to differentiate spontaneously in cell culture; 2) manipulating the cells so they will differentiate to form cells characteristic of the three germ layers; or 3) injecting the cells into a mouse with a suppressed immune system to test for the formation of a benign tumor called a teratoma. Since the mouse’s immune system is suppressed, the injected human stem cells are not rejected by the mouse immune system and scientists can observe growth and differentiation of the human stem cells. Teratomas typically contain a mixture of many differentiated or partly differentiated cell types—an indication that the embryonic stem cells are capable of differentiating into multiple cell types.

What are embryonic stem cells?

B. How are embryonic stem cells grown in the laboratory?
Growing cells in the laboratory is known as cell culture. Human embryonic stem cells are isolated by transferring the inner cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer. The mouse cells in the bottom of the culture dish provide the inner cell mass cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Researchers have devised ways to grow embryonic stem cells without mouse feeder cells. This is a significant scientific advance because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.
The process of generating an embryonic stem cell line is somewhat inefficient, so lines are not produced each time an inner cell mass is placed into a culture dish. However, if the plated inner cell mass cells survive, divide and multiply enough to crowd the dish, they are removed gently and plated into several fresh culture dishes. The process of re-plating or subculturing the cells is repeated many times and for many months. Each cycle of subculturing the cells is referred to as a passage. Once the cell line is established, the original cells yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic stem cell line. At any stage in the process, batches of cells can be frozen and shipped to other laboratories for further culture and experimentation.

What are embryonic stem cells?

A. What stages of early embryonic development are important for generating embryonic stem cells?

Embryonic stem cells, as their name suggests, are derived from embryos. Most embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro—in an in vitro fertilization clinic—and then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman's body. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocoel, a hollow cavity inside the blastocyst; and the inner cell mass, which is a group of cells at one end of the blastocoel that develop into the embryo proper.

What are the unique properties of all stem cells?

Stem cells differ from other kinds of cells in the body. All stem cells—regardless of their source—have three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types.
Stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells—which do not normally replicate themselves—stem cells may replicate many times, or proliferate. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal.
Scientists are trying to understand two fundamental properties of stem cells that relate to their long-term self-renewal:
1. why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most non-embryonic stem cells cannot; and
2. what are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?
Discovering the answers to these questions may make it possible to understand how cell proliferation is regulated during normal embryonic development or during the abnormal cell division that leads to cancer. Such information would also enable scientists to grow embryonic and non-embryonic stem cells more efficiently in the laboratory.
The specific factors and conditions that allow stem cells to remain unspecialized are of great interest to scientists. It has taken scientists many years of trial and error to learn to derive and maintain stem cells in the laboratory without them spontaneously differentiating into specific cell types. For example, it took two decades to learn how to grow human embryonic stem cells in the laboratory following the development of conditions for growing mouse stem cells. Therefore, understanding the signals in a mature organism that cause a stem cell population to proliferate and remain unspecialized until the cells are needed. Such information is critical for scientists to be able to grow large numbers of unspecialized stem cells in the laboratory for further experimentation.
Stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. For example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and it cannot carry oxygen molecules through the bloodstream (like a red blood cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.
Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. While differentiating, the cell usually goes through several stages, becoming more specialized at each step. Scientists are just beginning to understand the signals inside and outside cells that trigger each stem of the differentiation process. The internal signals are controlled by a cell's genes, which are interspersed across long strands of DNA, and carry coded instructions for all cellular structures and functions. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. The interaction of signals during differentiation causes the cell's DNA to acquire epigenetic marks that restrict DNA expression in the cell and can be passed on through cell division.
Many questions about stem cell differentiation remain. For example, are the internal and external signals for cell differentiation similar for all kinds of stem cells? Can specific sets of signals be identified that promote differentiation into specific cell types? Addressing these questions may lead scientists to find new ways to control stem cell differentiation in the laboratory, thereby growing cells or tissues that can be used for specific purposes such as cell-based therapies or drug screening.
Adult stem cells typically generate the cell types of the tissue in which they reside. For example, a blood-forming adult stem cell in the bone marrow normally gives rise to the many types of blood cells. It is generally accepted that a blood-forming cell in the bone marrow—which is called a hematopoietic stem cell—cannot give rise to the cells of a very different tissue, such as nerve cells in the brain. Experiments over the last several years have purported to show that stem cells from one tissue may give rise to cell types of a completely different tissue. This remains an area of great debate within the research community. This controversy demonstrates the challenges of studying adult stem cells and suggests that additional research using adult stem cells is necessary to understand their full potential as future therapies.

What are stem cells, and why are they important?

Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.
Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.
Until recently, scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic "somatic" or "adult" stem cells. The functions and characteristics of these cells will be explained in this document. Scientists discovered ways to derive embryonic stem cells from early mouse embryos nearly 30 years ago, in 1981. The detailed study of the biology of mouse stem cells led to the discovery, in 1998, of a method to derive stem cells from human embryos and grow the cells in the laboratory. These cells are called human embryonic stem cells. The embryos used in these studies were created for reproductive purposes through in vitro fertilization procedures. When they were no longer needed for that purpose, they were donated for research with the informed consent of the donor. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be "reprogrammed" genetically to assume a stem cell-like state. This new type of stem cell, called induced pluripotent stem cells (iPSCs), will be discussed in a later section of this document.
Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo, called a blastocyst, the inner cells give rise to the entire body of the organism, including all of the many specialized cell types and organs such as the heart, lung, skin, sperm, eggs and other tissues. In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of adult stem cells generate replacements for cells that are lost through normal wear and tear, injury, or disease.
Given their unique regenerative abilities, stem cells offer new potentials for treating diseases such as diabetes, and heart disease. However, much work remains to be done in the laboratory and the clinic to understand how to use these cells for cell-based therapies to treat disease, which is also referred to as regenerative or reparative medicine.
Laboratory studies of stem cells enable scientists to learn about the cells’ essential properties and what makes them different from specialized cell types. Scientists are already using stem cells in the laboratory to screen new drugs and to develop model systems to study normal growth and identify the causes of birth defects.
Research on stem cells continues to advance knowledge about how an organism develops from a single cell and how healthy cells replace damaged cells in adult organisms. Stem cell research is one of the most fascinating areas of contemporary biology, but, as with many expanding fields of scientific inquiry, research on stem cells raises scientific questions as rapidly as it generates new discoveries.

WHAT IS THE GOAL OF STEM CELL RESEARCH? Why don't we live forever?

Because we get sick?
Because we get old?
Because we get hurt and can't heal?
All of these are correct. Each one results from a failure of the body's ability to grow, maintain or repair itself - functions that depend on our stem cells.
In What are Some Different Types of Stem Cells?, we saw how stem cells form the basic building materials for the human body. This makes them good candidates for restoring tissues that have been damaged by injury or disease.
For decades, researchers have been studying the biology of stem cells to figure out how development works and to find new ways of treating health problems.

How would stem cell therapy work?
The goal of any stem cell therapy is to repair a damaged tissue that can't heal itself.
This might be accomplished by transplanting stem cells into the damaged area and directing them to grow new, healthy tissue.
It may also be possible to coax stem cells already in the body to work overtime and produce new tissue.
To date, researchers have found more success with the first method, stem cell transplants

New Biotech Breakthroughs that Will Change Medicine

1. Decay-Fighting Microbes
Bacteria living on teeth convert sugar into lactic acid, which erodes enamel and causes tooth decay. Florida-based company ONI BioPharma has engineered a new bacterial strain, called SMaRT, that cannot produce lactic acid—plus, it releases an antibiotic that kills the natural decay-causing strain. Dentists will only need to swab SMaRT, now in clinical trials, onto teeth once to keep them healthy for a lifetime.
2. Artificial Lymph Nodes
Scientists from Japan's RIKEN Institute have developed artificial versions of lymph nodes, organs that produce immune cells for fighting infections. Though they could one day replace diseased nodes, the artificial ones may initially be used as customized immune boosters. Doctors could fill the nodes with cells specifically geared to treat certain conditions, such as cancer or HIV.
3. Asthma Sensor
Asthma accounts for a quarter of all emergency room visits in the U.S., but a sensor developed at the University of Pittsburgh may finally cause that number to plummet. Inside the handheld device, a polymer-coated carbon nanotube—100,000 times thinner than a human hair—analyzes breath for minute amounts of nitric oxide, a gas that lungs produce prior to asthma attacks.
4. Cancer Spit Test
Forget biopsies—a device designed by researchers at the University of California-Los Angeles detects oral cancer from a single drop of saliva. Proteins that are associated with cancer cells react with dyes on the sensor, emitting fluorescent light that can be detected with a microscope. Engineer Chih-Ming Ho notes that the same principle could be applied to make saliva-based diagnostic tests for many diseases.
5. Biological Pacemaker
Electronic pacemakers save lives, but use hardware that eventually wears out. Now, researchers at several universities are developing a batteryless alternative: pacemaker genes expressed in stem cells that are injected into damaged regions of the heart. Better suited for physical exertion, biological pacemakers have been shown to bring slow canine hearts back up to speed without complications.

Thursday, August 19, 2010

DNA-Footprinting

DNA Footprinting was developed in 1977 and is an analytical procedure in molecular biology for identifying the specific sequence of DNA (the binding site) that binds to a particular protein. DNA Footprinting is most commonly performed on proteins that are thought to play some significant functional role such as gene regulation. This method can be performed on proteins which bind both double and single-stranded DNA. Additionally, DNA-binding proteins can be split into two groups, namely site-specific DNA-binding proteins and non-specific DNA–binding proteins.

DNA Footprinting uses a damaging agent such as a chemical reagent, radical or a nuclease that can cut or modify DNA at every base pair. However, where the ligand binds to DNA, the cleavage is restrained. DNA Footprinting discovers which specific parts of a DNA molecule have sites for specific proteins to attach to them. Using this technique, DNA that has first been in the presence of DNA-binding proteins and then exposed to a damaging agent, can be compared to DNA that was never exposed to the binding protein (and thus not protected against the damaging agent). The DNA sequence that is protected from cleaving can then be identified as the binding site.

DNA Footprinting can provide information that is, conceptually, much like fingerprinting in the sense that it can be used to identify a unique individual. DNA Footprinting can extract a banding pattern, or electropherogram, much like a bar code, that can identify a species or individual (some genes will be vary at the species level and others at the individual level)

Gene Therapy

Altered Genes 

Each of us carries about half a dozen defective genes. We remain blissfully unaware of this fact unless we, or one of our close relatives, are amongst the many millions who suffer from a genetic disease. About one in ten people has, or will develop at some later stage, an inherited genetic disorder, and approximately 2,800 specific conditions are known to be caused by defects (mutations) in just one of the patient's genes. Some single gene disorders are quite common - cystic fibrosis is found in one out of every 2,500 babies born in the Western World - and in total, diseases that can be traced to single gene defects account for about 5% of all admissions to children's hospitals. In the U.S. and Europe, there are exciting new programs to 'map' the entire human genome - all of our genes. This work will enable scientists and doctors to understand the genes that control all diseases to which the human race is prone, and hopefully develop new therapies to treat and predict diseases.

Diseases of Genetic Origin

Most of us do not suffer any harmful effects from our defective genes because we carry two copies of nearly all genes, one derived from our mother and the other from our father. The only exceptions to this rule are the genes found on the male sex chromosomes. Males have one X and one Y chromosome, the former from the mother and the latter from the father, so each cell has only one copy of the genes on these chromosomes. In the majority of cases, one normal gene is sufficient to avoid all the symptoms of disease. If the potentially harmful gene is recessive, then its normal counterpart will carry out all the tasks assigned to both. Only if we inherit from our parents two copies of the same recessive gene will a disease develop. On the other hand, if the gene is dominant, it alone can produce the disease, even if its counterpart is normal. Clearly only the children of a parent with the disease can be affected, and then on average only half the children will be affected. Huntington's chorea, a severe disease of the nervous system, which becomes apparent only in adulthood, is an example of a dominant genetic disease.
Finally, there are the X chromosome-linked genetic diseases. As males have only one copy of the genes from this chromosome, there are no others available to fulfill the defective gene's function. Examples of such diseases are Duchenne muscular dystrophy and, perhaps most well known of all, hemophilia.
Queen Victoria was a carrier of the defective gene responsible for hemophilia, and through her it was transmitted to the royal families of Russia, Spain, and Prussia. Minor cuts and bruises, which would do little harm to most people, can prove fatal to hemophiliacs, who lack the proteins (Factors VIII and IX) involved in the clotting of blood, which are coded for by the defective genes. Sadly, before these proteins were made available through genetic engineering, hemophiliacs were treated with proteins isolated from human blood. Some of this blood was contaminated with the AIDS virus, and has resulted in tragic consequences for many hemophiliacs. Use of genetically engineered proteins in therapeutic applications, rather than blood products, will avoid these problems in the future.
Not all defective genes necessarily produce detrimental effects, since the environment in which the gene operates is also of importance. A classic example of a genetic disease having a beneficial effect on survival is illustrated by the relationship between sickle-cell anemia and malaria. Only individuals having two copies of the sickle-cell gene, which produces a defective blood protein, suffer from the disease. Those with one sickle-cell gene and one normal gene are unaffected and, more importantly, are able to resist infection by malarial parasites. The clear advantage, in this case, of having one defective gene explains why this gene is common in populations in those areas of the world where malaria is endemic.

CLONING

The words 'cloning' and 'genetic engineering' are often used by people as though they mean the same thing. Well, they have an overlapping meaning that becomes clear when we look through history.

 "Genetic engineering, in its broadest definition, means to manipulate a species so that a particular trait is increased in the population.  A trait is how an organism looks or acts or what it does.   Brown eyes is a trait.   Flying in circles is a trait.  Climbing trees is a trait.

"The earliest forms of genetic engineering occurred on farms, where most people on earth lived at the time.  They managed to do this by selecting seeds from plants that maybe had more fruit production or tastier leaves than other plants of its type.


"They planted those seeds and grew plants that had more of the favorable traits.  Then they chose to save the seeds from the best of that lot to sow the next year.  So, year by year, the farmers produced better and better crops.   This type of activity probably has been going on since mankind first settled in villages and began making a life for themselves in one location, about 12,000 years ago!

 "The same sort of thing would have also happened with animals.  By eating the animals that didn't have favorable traits, like pulling a large load, and letting the animals with the favorable traits reproduce, herds and flocks would slowly develop more and more traits that humans found useful.  It was thousands of years before mankind figured out how plants and animals reproduce themselves.  With this knowledge, people could pollinate plants by hand or pen a pair of animals together in order to deliberately cause an increase in a favorable trait.




"It was only in the last 250 years that scientists began figuring out about chromosomes
and genes and the role they play in the way one generation passes its traits on to the next.  And its only been in the last 30 years that scientists have been able to cut out specific genes from one organism and put them in another.

 "It is this 30 year old technology that is described by the narrow definition of genetic engineering.  Mankind has long been able to have a deliberate impact on the world around him.  He now possesses the tools to deliberately impact himself.  Some people are afraid of what  might be done with that power.

"The word 'clone' was first used as a noun to describe a population of cells that reproduced themselves faithfully.  A clone produces cells that not only have the same chromosomes, but which turn on the same genes, turn off the same genes, and therefore look identical, act the same, and do the same things.

Molecular-Cloning

If scientists voted for the most essential biotechnology research tool, molecular cloning would likely win. Either directly or indirectly, molecular cloning has been the primary driving force of the biotechnology revolution and has made remarkable discoveries routine. The research findings made possible through molecular cloning include identifying, localizing and characterizing genes; creating genetic maps and sequencing entire genomes; associating genes with traits and determining the molecular basis of the trait. Molecular cloning involves inserting a new piece of DNA into a cell in such a way that it can be maintained, replicated and studied. To maintain the new DNA fragment, scientists insert it into a circular piece of DNA called a plasmid that protects the new fragment from the DNA degrading enzymes found in all cells. Because a piece of DNA is inserted, or recombined with, plasmid DNA, molecular cloning is a type of recombinant DNA technology.

The new DNA, now part of a recombinant molecule, replicates every time the cell divides. In molecular cloning, the word clone can refer to the new piece of DNA, the plasmid containing the new DNA and the collection of cells or organisms, such as bacteria, containing the new piece of DNA. Because cell division increases, or “amplifies,” the amount of available DNA, molecular cloning provides researchers with an unlimited amount of a specific piece of genetic material to manipulate and study.

Stem-Cell Technology

After animal cells differentiate into tissues and organs, some tissues retain a group of undifferentiated cells to replace that tissue’s damaged cells or replenish its supply of certain cells, such as red and white blood cells. When needed, these adult stem cells (ASCs) divide in two. One cell differentiates into the cell type the tissue needs for replenishment or replacement, and the other remains undifferentiated.
 

Embryonic stem cells (ESCs) have much greater plasticity than ASCs because they can differentiate into any cell type. Mouse embryonic stem cells were discovered and cultured in the late 1950s. The ESCs came from 12-dayold mouse embryo cells that were destined to become egg or sperm (germ cells) when the mouse matured. In 1981, researchers found another source of mouse ESCs with total developmental plasticity—cells taken from a 4-dayold mouse embryo.
In the late 1990s researchers found that human ESCs could be derived from the same two sources in humans:
primordial germ cells and the inner cell mass of 5-day-old embryos. Scientists also have been able to isolate pluripotent stem cells from human placentas donated following
normal, full-term pregnancies. Under certain culture conditions, these cells were transformed into cartilagelike
and fat-like tissue.
 

Maintaining cultures of ESCs and ASCs can provide answers to critical questions about cell differentiation: What factors determine the ultimate fate of unspecialized stem cells?
How plastic are adult stem cells? Could we convert an ASC into an ESC with the right combination of factors? Why do stem cells retain the potential to replicate indefinitely? Is the factor that allows continual proliferation of ESCs the same factor that causes uncontrolled proliferation of cancer cells?
If so, will transplanted ESCs cause cancer?
 

The answers to these questions and many more will determine the limits of the therapeutic potential of ESCs and ASCs. Only when we understand the precise mix of factors controlling proliferation and development will we be able to reprogram cells for therapeutic purposes. Using stem cell cultures, researchers have begun to elaborate the intricate and unique combination of environmental factors, molecular signals and internal genetic programming that decides a cell’s fate. Israeli scientists directed ESCs down specific developmental pathways by providing different growth factors. Others discovered that nerve stem cells require a dose of vitamin A to trigger differentiation into one specific type of nerve cell, but not another.