2011 January 06 Thursday
Single Mitochondrion Extracted From Cell

Mitochondria, organelles within cells that break down sugar to produce energy molecules for the rest of the cell, each have their own DNA for a subset of their genes. Some neuromuscular disorders are caused by mitochondrial DNA (mtDNA) mutations. A research team has now managed to develop tools that enable them to extract individual mitochondria from a cell.

Medical researchers who crave a means of exploring the genetic culprits behind a host of neuromuscular disorders may have just had their wish granted by a team working at the National Institute of Standards and Technology (NIST), where scientists have performed surgery on single cells to extract and examine their mitochondria.

Why is this useful? Well, if mutations are causing problems a natural question is whether all mtDNA copies in each cell have the same mutation. If each one can be isolated and separately sequenced then their sequences can be compared. Do all mtDNA in a cell share the same sequence? Do all cells have the same mtDNA sequence?

Lasers and a tiny pipette did the trick.

The research team, which also includes scientists from Gettysburg College, has potentially solved this problem by realizing that several devices and techniques can be used together to extract a single mitochondrion from a cell that possesses a genetic mutation. They employed a method** previously used to extract single chromosomes from isolated rice cells where a laser pulse makes an incision in a cell's outer membrane. Another laser is used as a "tweezer" to isolate a mitochondrion, which then can be extracted by a tiny pipette whose tip is less than a micrometer wide.

What would also be useful: the ability to deliver mitochondria into a cell. For example, suppose a woman has a mitochondrial mutation that is problematic and yet she wants to make babies. It would be handy to be able to replace the mitochondria in one of her eggs with mitochondria that do not have the mutation.

By Randall Parker    2011 January 06 10:11 PM   Entry Permalink | Comments (0)
2010 December 22 Wednesday
Researchers Separate Chromosomes For Sequencing

An article in Technology Review reports on advances made in separating chromosomes so they can be individually sequenced.

Now two teams have devised ways to determine these groupings—known as the haplotype—in an individual. Stephen Quake and collaborators at Stanford University developed a way to physically separate the chromosome pairs and sequence each strand of DNA individually. Jay Shendure and colleagues at the University of Washington in Seattle sequenced DNA from single chromosomes in specially selected pools and used this information to piece together the genome. Both projects were published this week in Nature Biotechnology.

If each chromosome in a cell can be separated out and individually sequenced then one could do the same to parents and children. With that information it will be easy to figure out exactly which chromosomes each child inherited.

This gets especially interesting when thinking about reproduction. If each person can know which genetic variants they have on which chromosome then couples could think about the ramifications of all the possible combinations of their chromosomes they could give to their offspring.

We still need the technical means to choose chromosomes to assemble the more desired chromosomes from each parent into an embryonic cell to start a pregnancy. Given that capability the rate of human evolution will accelerate by orders of magnitude.

The rate of sequencing of full human genomes is rising by orders of magnitude. We need a flood of genetic data needed to figure out what all the genetic variants mean. That flood is starting to happen.

In the last year, the number of sequenced, published genomes has shot up from two or three to approximately nine, with another 40 or so genomes sequenced but not yet published. "While the numbers are still small numbers, we are starting to put this research into the real disease context and get something out of it," says Jay Shendure, a geneticist at the University of Washington in Seattle, and a TR35 winner in 2006.

Given an in-depth understanding of the human genome and the means to choose chromosomes for offspring human evolution will accelerate by orders of magnitude. It is only a matter of when, not if. The knowledge is coming over the next 10-20 years. The technology for choosing between embryos with in vitro fertilization will enable some acceleration of evolution. But the ability to choose chromosomes will bump up the rate of evolution by orders of magnitude more.

By Randall Parker    2010 December 22 08:12 AM   Entry Permalink | Comments (1)
2010 January 13 Wednesday
Nanowires Poke Molecules Into Cells

Nanowires can deliver drugs and other compounds into cells grown in culture

Many experiments in biology rely on manipulating cells: adding a gene, protein, or other molecule, for instance, to study its effects on the cell. But getting a molecule into a cell is much like breaking into a fortress; it often relies on biological tricks such as infecting a cell with a virus or attaching a protein to another one that will sneak it through the cell's membrane. Many of these methods are specific to certain types of cells and only work with specific molecules. A paper in this week's Proceedings of the National Academy of Sciences offers a surprisingly simple and direct alternative: using nanowires as needles to poke molecules into cells.

This involves growing cells on a bed of nanowires. The nanowires can poke into the cells and then release molecules once they are inside. This will enable rapid screening of how many different kinds of molecules affect the behavior of cells.

I can imagine something like these nanowires used to deliver gene therapy into cells. We still need better ways to deliver gene therapy. While this technique might help with gene therapy in vitro it does not appear to provide a better way to deliver genes into cells in the body (i.e. in vivo). We need great in vivo gene therapy delivery mechanisms especially in order to rejuvenate the brain. Gotta deliver new genetic programming into old cells that hold the memories and skills accumulation of our lives.

By Randall Parker    2010 January 13 10:31 PM   Entry Permalink | Comments (1)
2006 August 21 Monday
Electric Tweezers Move Microscopic Objects

Yet another sign that desktop laboratories will become cheaper, more widely accessible, more automatable:

PHILADELPHIA -- The ability to sort cells or manipulate microscopic particles could soon be in the hands of small laboratories, high schools and amateur scientists, thanks to researchers at the University of Pennsylvania School of Engineering and Applied Science. They have created a device, called "electric tweezers," which can manipulate and move almost any object seen on a simple microscope slide.

The research was led by graduate student Brian Edwards, with the help of his advisor Nader Engheta, professor, and Stephane Evoy, adjunct assistant professor, both of Penn's Electrical and Systems Engineering Department. While devices with similar functionality using lasers exist, they often cost upwards of a quarter-million dollars. Edwards' device performs some of the same tasks as laser tweezers, yet at a price anticipated to be in the same range as a high-end desktop computer.

"The tweezers create an electric field that you can use to manipulate almost any object on a microscopic scale. It has the potential of being a powerful tool for research," said Edwards, a doctoral candidate in Penn's Electrical and Systems Engineering Department. "I would prefer not to put a limit on the type of tasks that can be done with it, but I hope it will find uses in anything from picking an individual cell out of a culture to fabricating circuits."

All it would take to use electric tweezers is a computer and a microscope. The tweezers' action occurs on a common glass microscope slide embedded with five electrodes. These electrodes create an electric field that can be used to push, pull, move and spin a selected object in any direction without actual physical contact. Using software Edwards developed, an operator can select an individual object from a microscope image on a computer screen.

"Different types of particles respond differently to different frequencies in the electric field," Edwards said. "Once you lock onto the object of interest you can move it however you like."

The electric tweezers take advantage of the phenomenon known as dielectrophoresis, where electric fields impart a force upon a neutral particle. In essence, the object that is selected surfs atop the hills and valleys created by subtly changing the electric field. The principle works best on the microscopic scale, which makes it ideal for this application.

Biotechnology is headed down the same path as electronic technology. Computers are accelerating the advance of biotechnology and processes developed to make semiconductors help to push microfluidics technology forward. The general trend is going to be toward smaller, cheaper, and more automated lab instruments.

By Randall Parker    2006 August 21 10:21 PM   Entry Permalink | Comments (0)
2006 May 25 Thursday
Protein Target Found To Block Muscle Atrophy

A drug and a gene therapy can block muscle atrophy in mice which get little exercise.

Researchers in Purdue University's School of Veterinary Medicine have discovered genetic and drug-treatment methods to arrest the type of muscle atrophy often caused by muscle disuse, as well as aging and diseases such as cancer.

The findings might eventually benefit people who have been injured or suffer from diseases that cause them to be bedridden and lose muscle mass, or sometimes limbs, due to atrophy, said Amber Pond, a research scientist in the school's Department of Basic Medical Sciences.

...

"We've found a chemical 'switch' in the body that allows us to turn atrophy on, and, from that, we also have learned how to turn atrophy off."

A protein called Merg1a plays a key role in allowing or blocking muscle atrophy and atrophy can be blocked by an antihistamine which targets Merg1a and also by gene therpy.

The research team found atrophy of skeletal muscle in mice could be inhibited with both gene therapy and drug treatment using astemizole (as-TEM-uh-zole), an antihistamine. This new insight has potential in many different areas of research, Pond said.

"We have discovered a direct link between atrophy and a protein in the skeletal muscle," Pond said. "This led us to develop methods that would block the protein's ability to cause atrophy. Through drug treatment, we were able to block atrophy, allowing muscle to retain 97 percent of its original fiber size in the face of atrophy."

Astemizole, which was withdrawn from the market in 2000 because of its potential to cause serious cardiovascular problems, wouldn't be suitable for use in humans, Pond said. The drug can be used in mice because it doesn't affect their hearts to the same extent.

While the drug used in the experiment isn't suitable for human use this discovery points toward a direction for drug development to prevent muscle atrophy with cancer, age, or low exercise.

A mutant form of Merg1a inserted with gene therapy prevented muscle atrophy in low exercise mice.

This method allowed the scientists to demonstrate the effects of skeletal muscle atrophy and investigate reasons for the link with the Merg1a protein. The Merg1a protein is a channel that normally passes a small electrical current across the cell.

The researchers implanted a gene into the skeletal muscle that resulted in a mutant form of this protein that combines with the normal protein and stops the current. The researchers found that the mutant protein would inhibit atrophy in mice whose ability to use their back legs was limited.

Because gene therapy is not yet a practical treatment option in humans, the researchers decided to go a step further and stop the function of the protein with astemizole, which is a known "Merg1a channel blocker." The researchers found that the drug produced basically the same results as the gene therapy. In fact, muscle size increased in mice in the group that were given the drug without any other treatment.

"We are now looking at the differences in the structure of the heart and the skeleton to give us clues on how to specifically target muscles without the cardiac side effects," Pond said.

A lot of people would love to have the effects of exercise last longer. Also, a method to avoid muscle atrophy with age would have very wide appeal.

By Randall Parker    2006 May 25 12:19 AM   Entry Permalink | Comments (4)
2005 October 28 Friday
Implantable Remote Controlled Surgical Robot Developed

University of Nebraska researchers have developed a 3 inch (8 centimeters) insertable remote controlled robot for abdomen surgery.

But, these tiny-wheeled robots – slipped into the abdomen and controlled by surgeons hundreds of kilometers away – may be giants in saving the lives of roadside accident victims and soldiers injured on the battlefield.

Each camera-carrying robot -- the width of a lipstick case -- would illuminate the patient’s abdomen, beam back video images and carry different tools to help surgeons stop internal bleeding by clamping, clotting or cauterizing wounds.

Sound far-fetched? Not for physicians and engineers at the University of Nebraska Medical Center and University of Nebraska-Lincoln, who already are turning the sci-fi idea into reality with a handful of miniature prototypes.

“We want to be the Microsoft leader in this technology and be the state that changes the way surgery is done,” said Shane Farritor, Ph.D., associate professor in the Department of Mechanical Engineering in UNL’s College of Engineering and Technology.

“This work has the potential to completely change the minimally invasive surgery landscape,” said Dmitry Oleynikov, M.D., director of education and training for the minimally invasive and computer-assisted surgery initiative. “This is just the start of things to come regarding robotic devices at work inside the body during surgery.”

So when will surgery by hands-on surgeons become less common than surgery by robots that are controlled by surgeons? 20 years? 30 year? When will surgeon-controlled robots be replaced by totally automated robots?

This approach provides greater control and more views than existing laparoscopic techniques.

It’s a stark contrast to existing laparoscopic techniques, which allow surgeons to perform operations through small incisions. The benefits of laparoscopy are limited to less complex procedures, however, because of losses in imaging and dexterity compared to conventional surgery.

“These remotely controlled in vivo robots provide the surgeon with an enhanced field of view from arbitrary angles, as well as provide dexterous manipulators not constrained by small incisions in the abdominal wall,” Dr. Oleynikov said.

In fact, the view is better than the naked eye, he said, because the in-color pictures from the roaming robots are magnified 10x.

Future remote use applications include space, battlefield, and civilian emergencies.

On the battlefield, these tiny soldiers can be inserted into wounds and allow remote surgeons to determine how critical the injury is and what immediate steps can be taken to ensure survival.

The UNMC and UNL team also plans to soon test a final prototype of a mobile biopsy robot designed to take samples of tissue. In addition, the design team is making modified robots that can be inserted into the stomach cavity through the esophagus.

The 3-inch long, aluminum-cased robots contain gears, motors, lenses, camera chips and electrical boards. “Three inches seems to be our limit at the moment because of the electrical components we use,” said designer Mark Rentschler, a Ph.D. candidate in biomedical engineering at UNL. “If we were to make 1,000 robots we would be able to afford customized electrical components that would reduce the size of the robot by half.”

The design team said initially the mini-robots will be single-use devices, although they eventually may be able to be sterilized for multiple use.

The group intends to create a local, spin-off company and then seek FDA approval of the devices, which would be applicable for any laparoscopic or minimally invasive surgery – from gall bladder to hernia repair.

NASA will begin trials next spring with an astronaut in a submarine off of Florida. The scientists hope to begin clinical trials with humans within a year in the UK.

One can also imagine an insertable stem cell incubator that would continually produce stem cells aimed at an especially damaged part of the body. Or how about an insertable robot surgeon that stays in the body for days and weeks to gradually reshape damaged tissue with a combination of a series of small surgical modifications, drug delivery, and stem cell delivery? In the longer run nanobots will do a lot of that work. But before nanobots become practical more conventional miniaturized robots will do a lot of repair work.

By Randall Parker    2005 October 28 10:34 AM   Entry Permalink | Comments (8)
2003 June 11 Wednesday
Selective Blocking Of Mutant Genes Demonstrated

University of Iowa graduate student Victor Miller and other researchers have demonstrated the ability to selectively turn off a copy of a gene that differs by only a single nucleotide from other copies of the same gene in the same cell.

Turning off a mutant gene while keeping the normal gene active would be particularly useful in therapies aimed at treating so-called dominantly inherited diseases. In these diseases, a single mutant copy of a gene inherited from either parent dominates the normal gene by producing a protein that is toxic to cells. Thus, a successful therapy must remove or suppress the disease-gene rather than simply add a corrected version. At the same time, the normal gene may be essential, so it is important to be able to silence the disease-causing gene without affecting the normal copy. Many neurodegenerative conditions, including Huntington's disease (HD), are dominantly inherited. The HD gene also is an example of a normal gene that appears to be essential for normal function.

Working in cell culture, the UI researchers used the relatively new technology known as RNA interference to silence a mutant gene that causes the neurodegenerative condition called Machado-Joseph disease (or Spinocerebellar Ataxia Type 3), while leaving the normal gene alone.

Machado-Joseph disease (MJD), Huntington's disease and at least seven other neurodegenerative disorders all are caused by the same type of genetic mutation. The genetic defect in these diseases produces a mutated protein with an abnormally long stretch of a repeated amino acid. The mutant protein in each of these conditions is prone to clump together, forming aggregates, which appear to damage brain tissue. Other neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, also are characterized by a tendency for proteins to misfold or clump in the brain. The UI team studies Machado-Joseph disease because it is a good model for investigating these types of neurodegenerative diseases.

Initial attempts to silence the mutant MJD gene by targeting the RNA interference to the repeat-expansion mutation failed. So the UI researchers focused on a single sequence difference, also known as a single nucleotide polymorphism (SNP), which occurs just next to the mutated sequence in about 70 percent of mutant MJD genes.

"When we tried to target the mutation itself, the interfering RNA was not able to distinguish the mutant gene from the normal gene and both copies were suppressed," said Henry Paulson, M.D., Ph.D., UI assistant professor of neurology and principle investigator of the study. "Then we noticed that there was a single nucleotide polymorphism in the mutant MJD gene that comes right after the mutation in most cases. We targeted that single nucleotide variation with RNA interference and that approach was able to distinguish the mutant from the normal and only knock down the mutant gene."

Paulson added that the discovery that RNA interference could distinguish between genes on the basis of a single nucleotide polymorphism was very exciting because every person's DNA differs mostly on the basis of these unique single letter variations in the genetic code. Thus it might be possible to use RNA interference to target unique single nucleotide polymorphisms associated with specific genes in order to manipulate those genes.

"Even when one cannot target a disease-causing mutation, it may still be possible to target the mutant gene on the basis of a SNP associated with that gene," Paulson said.

The research team also used RNA interference to target an actual disease-causing mutation due to a single base pair change in a gene. Tau is an important cellular protein that is mutated in some inherited dementias that are somewhat similar to Alzheimer's disease. The UI researchers directed RNA interference against a specific mutation in the Tau gene that is known to cause dementia in people. Again, the approach was successful in silencing only the mutant gene and not the normal gene.

This result is important because it is harder to replace a gene in a cell than it is to add another gene. Picture a cell that has two different variants of the same gene. Most cells have at least 2 copies of almost every gene (a notable exception being the genes that are on the X chromosome in males). Some people have genetic diseases that are the result of a dominant mutation. When a harmful mutation is dominant only one of the two copies of a gene has to have the mutation in order for the mutation to have harmful effects. The ability to effectively turn off the expression of just the harmful copy would be very valuable. This report provides evidence that even a point mutation of a single nucleotide in the DNA sequence of a gene (called a Single Nucleotide Polymorphism or SNP) provides enough of a difference to be targetted by RNA interference therapies.

To make RNA interference useful for many genetic disorders what is needed is a gene therapy that will cause copies of the interfering RNA sequence to be present in cells for a long time. To do that what is needed is the ability to add DNA to a cell that would persist and continually be used to make interfering RNA. The big enabling technologies needed are mini-chromosomes and a mechanism for delivering mini-chromsomes into large numbers of cells of a target cell type (e.g. into all the neurons in a brain). If a very small mini-chromosome could be added to a cell that expressed a sequence that could do RNA interference against the harmful variation of a gene then it would be possible to prevent the harmful gene from causing problems.

By Randall Parker    2003 June 11 03:01 PM   Entry Permalink | Comments (0)
2003 June 09 Monday
Epigenetic Factors Reverse Nucleus Cancer State

Transfer of a cell nucleus from a cancer cell to a normal cell does not turn the normal cell into a cancer cell.

Nuclei removed from mouse brain tumor cells and transplanted into mouse eggs whose own nuclei have been removed, give rise to cloned embryos with normal tissues, even though the mutations causing the cancer are still present. This research, from scientists at St. Jude Children's Research Hospital, appears in the June 1 issue of Cancer Research.

The finding demonstrates that the cancerous state can be reversed by reprogramming the genetic material underlying the cancer, according to James Morgan, Ph.D., a member of the St. Jude Department of Developmental Neurobiology, and lead author of the study. The findings also indicate that genetic mutations alone are not always sufficient to cause a cell to become cancerous.

“Specifically, it shows that so-called epigenetic factors are key elements in the development and maintenance of tumors,” Morgan said.

Epigenetic factors are those that influence the cell’s behavior. Examples include environmental effects and chemical modification.

“The concept of epigenetic factors having a role in cancer is already largely accepted,” Morgan said. “In fact, it’s already known that epigenetic alterations of chromosomes can cause certain rare forms of cancer. And some anti-cancer agents actually target epigenetic changes. But this is the first formal proof of the theory in a living animal.”

Unlike mutations, epigenetic modifications of DNA are potentially reversible molecular events that cause changes in gene expression. Some genes that help prevent the development of cancer (e.g., tumor suppressor genes) can be targets of epigenetic factors. The inactivation of such a gene might make the DNA more vulnerable to developing a cancer-causing mutation.

A cell is an incredibly complex state machine. The transition of a cell into a cancerous state may require (at least in some cases) more than just a set of mutations in the nucleus. The challenge is going to be to figure out what it is about an egg cell that allows it to turn the nucleus from a cancerous cell back into a non-cancerous state.

The epigenetic state that is producing this effect might be enzymes that methylate (attach methyl groups to) nuclear DNA. Or it might be molecules that bind to DNA at sites in the nucleus where binding will shut down replication. Or possibly the key might not be something that is in the egg cells. The key could be that the egg cell cytoplasm is missing some compounds that are necessary to maintain rapid cell division. Those compounds might even be essential for telling the nucleus to make enzymes that make more of those compounds. There are just a lot of imaginable ways that the epigenetic state difference might be working to convert a cancerous nucleus back into a non-cancerous state.

Note that they transferred the nuclei from cancer cells into eggs, not into regular cells. An egg has only half the normal amount of DNA that a normal cell has since it has only one member of each pair of chromosomes. An egg's epigenetic state is very different from that of adult cells. It is so different that it is possible in some species to put adult non-cancerous cell nucleuses into eggs to make embryos. This doesn't work every time but it can be used to clone animals.

It would be interesting to know whether this transfer of a cancer cell nucleus into a different type of cell has ever been tried with non-egg cells as targets.

Update: Providing what may be a clue to how a nucleus could be shifted back to a non-cancerous state, a sey of recent papers point to an important role for methylation in cancer development. (free registration at The Scientist site required)

Minna's team has gone on to characterize RASSF1 expression in more than 1,000 tumor samples. "I would say that after p53, it's the most frequently inactivated tumor suppressor gene," says Minna. Some already have begun to link RASSF1A status with prognosis. "Several studies have now shown that the presence of RASSF1A methylation confers worse prognosis on non-small-cell lung cancer patients," he says.5

What will be interesting to see is whether epigenetic changes such as methylation are being caused by mutations elsewhere in the genome.

By Randall Parker    2003 June 09 02:58 AM   Entry Permalink | Comments (2)
2003 February 24 Monday
Pluripotent Stem Cells Made From Blood Cells

Adult monocyte white blood cells can be converted into stem cells that can become many other cell types.

The particularly powerful – and very scarce – flexible forms of stem cells needed for medical research and treatment may now be both plentiful and simple to produce, with a new technology developed at the U.S. Department of Energy’s Argonne National Laboratory – and the source is as close as your own bloodstream.

These flexible stem cells, able to morph into a variety of cell types, are called “pluripotent,” and before this Argonne research, they have been found only in fetal tissue, which is limited, and in bone marrow, which is difficult to collect. Pluripotent stem cells are important because they can generate all types of tissues found in the body, and the Argonne-developed technology can produce them from adult blood cells.

The finding may eventually offer researchers a practical alternative to the use of embryonic stem cells for research, drug discovery, and transplantation.

Argonne scientist Eliezer Huberman and his colleagues, Yong Zhao and David Greene, examined adult monocytes, a type of white blood cells that act as precursors to macrophages. The researchers found that when monocytes were exposed to a growth factor, they created a set of pluripotent stem cells. After cultivating the stem cells, the scientists were able to make the cells “differentiate” into nerve, liver, and immune system tissue by delivering more growth factors.

“Because of its great promise in medicine, I’m prouder of this work than of anything else I’ve done,” Huberman said.

The research is being published in the Proceedings of the National Academy of Sciences.

Storing the precursor cells in liquid nitrogen had no effect on their differentiation later. Because monocytes can be easily gathered from a patient's own blood supply, the researchers suggest that treating disease with a genetic match to prevent rejection may be possible in the future.

This means that the material should produce valuable candidates for transplantation therapy, useful to replenish immune cells that have been eradicated by cancer therapy or to replace neuronal tissue damaged during spinal cord injury, stroke, Alzheimer’s or Parkinson’s disease.

Funding for the research is from the National Institutes of Health. The researchers have applied for a patent on the new technology.

This is a very exciting development. By avoiding cloning or the use of embryos or fetal tissue this technique may not provoke as much opposition on ethical grounds. There is still a possibility that some will ethically object. If these cells turn out to be capable of developing into a fetus some might argue that application of the growth factor is essentially creating an embryo.

It is premature to conclude that these cells are pluripotent and as fully useful as embryonic stem cells. Other labs are going to have to confirm that the cells can become many more cell types. But if the cells can do that then the next question that needs to be asked is whether they are as youthful as stem cells taken from an embryo or umbilical cord. Tests need to be done to measure telomere sizes, ability to go thru many cell divisions, speed of cell division, and other indicators.

By Randall Parker    2003 February 24 12:47 PM   Entry Permalink | Comments (0)
2003 February 21 Friday
Human Embryonic Stem Cell Gene Insertion and Deletion

Scientists at U Wisc Madison have developed a technique for humans to add or delete genes from embryonic cells. (bold emphases added)

MADISON - The technique that helped revolutionize modern biology by making the mouse a crucible of genetic manipulation and a window to human disease has been extended to human embryonic stem (ES) cells.

In a study published today (Feb. 10) in the online editions of the journal Nature Biotechnology, a team of scientists from the University of Wisconsin-Madison reports that it has developed methods for recombining segments of DNA within stem cells.

By bringing to bear the technique, known in scientific parlance as homologous recombination, on DNA in human embryonic stem cells, it is now possible to manipulate any part of the human genome to study gene function and mimic human disease in the laboratory dish.

"Indeed, homologous recombination is one of the essential techniques necessary for human ES cells to fulfill their promise as a basic research tool and has important implications for ES cell-based transplantation and gene therapies," write Wisconsin researchers Thomas P. Zwaka and James A, Thomson, the authors of the new study.

The technique has long been used in the mouse and is best known in recent years for its use to generate mice whose genomes have been modified by eliminating one or more genes. Known as 'knockouts,' genetically altered mice have become tremendously important for the study of gene function in mammals, and have been used to explore everything from the underlying mechanisms of obesity and other conditions to the pinpointing of genes that underpin many different diseases.

Significant differences between mouse and human embryonic stem cells have, until now, hampered the application of the technique to human ES cells, according to Zwaka, the lead author of the Nature Biotechnology report and a research scientist working in the laboratory of James Thomson. Thomson was the first to isolate and culture human embryonic stem cells nearly five years ago.

"This is a big benefit for the human ES cell field," Zwaka said. "It means we can simulate all kinds of gene-based diseases in the lab - almost all of them."

To demonstrate, the team led by Zwaka and Thomson were able to remove from the human genome the single gene that causes a rare genetic syndrome known as Lesch-Nyhan, a condition that causes an enzyme deficiency and manifests itself in its victims through self-mutilating behavior such as lip and finger biting and head banging.

The study of genes derived from human ES cells, as opposed to those found in mice, is important because, while there are many genetic similarities between mice and humans, they are not identical. There are human genes that differ in clinically significant ways from the corresponding mouse genes, said Zwaka. The gene that codes for Lesch-Nyhan is such a gene, as mice that do not have the enzyme do not exhibit the dramatic symptoms of the disease found in humans whose genes do not make the enzyme.

Another key aspect of the new work is that it may speed the effort to produce cells that can be used therapeutically. Much of the hype and promise of stem cells has centered on their potential to differentiate into all of the 220 kinds of cells found in the human body. If scientists can guide stem cells - which begin life as blank slates - down developmental pathways to become neurons, heart cells, blood cells or any other kind of cell, medicine may have access to an unlimited supply of tissues and cells that can be used to treat cell-based diseases like Parkinson's, diabetes, or heart disease. Through genetic manipulation, 'marker' genes can now be inserted into the DNA of stem cells destined for a particular developmental fate. The presence or absence of the gene would help clinicians sort cells for therapy.

"Such 'knock-ins' will be useful to purify a specific ES-cell derived cell type from a mixed population," Zwaka said. "It's all about cell lineages. You'll want dopamine neurons. You'll want heart cells. We think this technique will be important for getting us to that point."

Genetic manipulation of stem cells destined for therapeutic use may also be a route to avoiding transplant medicine's biggest pitfall: overcoming the immune system's reaction to foreign cells or tissues. When tissues or organs are transplanted into humans now, drugs are administered to suppress the immune system and patients often need lifelong treatment to prevent the tissue from being rejected.

Through genetic manipulation, it may be possible to mask cells in such a way that the immune system does not recognize them as foreign tissue.

This press release is as notable for what it doesn't say as for what it does say. First lets review what it does say the technique will be useful for.

Yes, this technique will be useful for doing cloning to create cell lines that have knock-outs of genes in order to study the effects of eliminating individual genes. This is routinely done with mouse cell lines. It can even be used with reproductive cloning to find out whether a mouse can still live without a gene and to see what effects the absence of the gene has on whole organisms. Of course it is unlikely (at least in Western countries) that scientists are going to do reproductive cloning on humans with gene knock-outs to discover what effects a gene knock-out has on full human organisms.

The technique will probably be useful in helping to guide cellular differentiation and to solve immuno-compatibility problems in order to create replacement organs.

The press release doesn't mention the possibility that this technique will also probably be useful for the creation of non-embryonic stem cell lines in order to do replenishment of non-embryonic stem cell reservoirs. That will be useful for treating some genetic diseases (e.g. sickle cell anemia) and some types of cancer (e.g. leukemia). But the biggest benefit to come from making non-embryonic stem cells is as a rejuvenation therapy to partially reverse aging.

The biggest potential use of this technique that the press release doesn't mention is like the huge elephant in the room that everyone pretends not to notice: This technique will be useful for human germ line genetic engineering for the purpose of creating genetically engineered human offspring. The ability to delete and insert genes means the ability to replace one version of a gene with a different version of the same gene. It also means the ability to add new genes. Also, additional copies of existing genes could be added in order to get more expression of those genes in the embryo, child, or adult human.

A technique to change embryonic stem cell genes is unlikely to be limited only to embryonic stem cells produced in a single way. It seems likely that the technique will work on embryos produced by cloning or by in vitro fertilization of an egg or by regular sexual reproduction where the embryonic cells would be removed from a woman's womb. Therefore regardless of how a viable embryo is created it will be possible to do genetic engineering to it before it develops into a fetus and baby.

By Randall Parker    2003 February 21 03:24 PM   Entry Permalink | Comments (6)
2003 January 22 Wednesday
Modified Printers Used For Tissue Engineering

Scientists at the Medical University of South Carolina are using inkjet printers to lay down cells and gels to make 3 dimensional cell structures.

Three-dimensional tubes of living tissue have been printed using modified desktop printers filled with suspensions of cells instead of ink. The work is a first step towards printing complex tissues or even entire organs.

"This could have the same kind of impact that Gutenberg's press did," claims tissue engineer Vladimir Mironov of the Medical University of South Carolina.

One enabling technology for this work came from Thomas Boland of Clemson University who developed the idea of printing biomaterials on surfaces.

1. Protein Printing
The research involves deposition of proteins in patterns or arrays using the protein printer, a device developed in the laboratory. Protein printing allows high throughput, fully automated deposition of a variety of biomolecules such as DNA , proteins, antibodies or drugs onto polymeric supports such as petri-dishes or tissue engineering scaffolds. Currently, the device is used to analyze 300,000 potential anti cancer drugs for their ability to prevent angiogenesis.

2. Cell Printing
Cell printing is the extension of protein printing to entire cells. The cell printer developed in the laboratory is fully automated and allows to deposit live cells with 500 nm precision on to supports such as tissue engineering scaffolds. Current research includes the deposition of enothelial cells for in vitro tube formation, single cell microculture and single cancer cell characterization.

An important enabling technology for this work is the thermoreversible gel which is also delivered by a printer cartridge over each cell layer to provide a structure to allow build-up of a 3 dimensional structure.

Called a stimuli-sensitive polymer, the material is designed to change immediately from a liquid into a gel in response to stimulus, such as an increase in temperature. This feature would enable physicians to inject the mixture of the polymer and a medicinal solution directly into a specific target in the body, where it would warm and instantly gel.

"Stimuli-sensitive gels show promise for the effective treatment of inoperable tumors," said Anna Gutowska, senior research scientist at PNNL and lead developer of the gel. "While much more research remains to be done before this becomes an accepted medical procedure, we are very excited about its potential."

Gutowska has spent many years developing biocompatible gels for drug delivery, cartilage repair and other medical applications. This latest work appears to be an outgrowth of her previous collaboration with MUSC researchers to use a gel as a scaffolding for the growth of cartilage.

In related research, PNNL is collaborating with the Medical University of South Carolina to test a biodegradable version of the polymer gel to support repair of articular cartilage—the durable type of cartilage that provides cushion between knee joints and other joints in the body.

Once injured, articular cartilage doesn't heal well, or typically at all on its own. Consequently, more than one million cartilage repair surgeries are conducted annually. However, there are limitations to the effectiveness of these surgeries because physicians have been unable to spur growth of articular cartilage inside the body.

To try to encourage growth and healing, cartilage cells, called chondrocytes, are extracted from a different site within the body for cultivation in the laboratory. Not only does this create another defect at the removal site, but physicians have been unable to cultivate chondrocytes with all the properties required to generate articular cartilage. Rather, a weaker, less durable type called fibrocartilage forms.

Through a two-year, DOE-funded project, Gutowska and collaborators at the Medical University of South Carolina are developing two components to support the successful repair of articular cartilage. The first is a three-dimensional cell culture system to support the in-laboratory growth of chondrocytes that retain the properties necessary for articular cartilage repair. A patent recently was issued for this technology.

The second component is a biodegradable polymer gel that can be injected into the defect to serve as a temporary synthetic "scaffold" to support growth of the injected chondrocytes. Testing of the biodegradable gel currently is taking place at the Medical University of South Carolina.

The idea of using common inkjet printers for laying down biomaterials and even living cells demonstrates how advances in other technological fields provide mature technologies for use in bioengineering. Also, the development of the thermosensitive biocompatible gel demonstrates that bioengineering involves a lot more than just the understanding and manipulation of cells.

By Randall Parker    2003 January 22 07:59 PM   Entry Permalink | Comments (3)
2002 December 18 Wednesday
Radio Remote Controlled Bacteria

Consider the cloak and dagger possibilities for when bacteria will become controllable by radio waves.

Only millionths of a millimetre across, the gold nanoparticle acts as an antenna, harvesting energy from a radio-frequency electromagnetic field. This energy breaks up the enzyme, rendering it useless. When the field is switched off, the parts of the enzyme re-assemble of their own accord.

This is grist for science fiction and spy TV show and movie plots. Imagine someone who could be blackmailed by the threat of activating dormant bacteria in their body. "Mr. Bond, if you do not cooperate with us immediately I will unleash the bubonic plague bacteria in you with a flick of this button." Of course Bond would have a radio cigarette lighter signal jammer that Q gave him. He could have secretly seduced the fiendish bad guy's equally bad girlfriend the night before and unknowingly infected her with the bacteria too. When the bad guy flicks the activation signal she'd collapse on the balcony and they'd both see it happen thru the plate glass window. The bad guy would run at him in a rage and Bond would deftly send him thru the plate glass window and over the balcony to his death.

The scaled up mega-disaster version would involve a dormant bacteria that had infected most of a country's population. Terrorists would threaten to kill them all unless assorted demands were met.

By Randall Parker    2002 December 18 02:21 PM   Entry Permalink | Comments (3)
2002 October 25 Friday
Chromosome Twin Sisters Used To Repair DNA Damage

Each cell gets on average 20,000 mutations per day on its chromosomes. There are a number of repair mechanisms for dealing with this damage. These Israeli scientists have demonstrated a long hypothesized repair mechanism. Since chromosomes come in pairs it is possible for a cell to repair one chromosome by copying the equivalent section of the other member of its pair:

The other last-resort repair system was hypothesized by scientists in the 1960s yet was never proved until the current study. This system, which relies on the help of “sister chromosomes,” enables the cell to repair genetic damage without the risk of creating mutations. (During the process of cell division, each chromosome - the structure in the nucleus that contains DNA - gives rise to two identical “sister” chromosomes. These move on to the two separate cells created from the dividing cell.)

According to this theory, if one of the sister chromosomes is damaged, the other can serve as a back-up system of sorts. The damaged genetic information can be restored precisely using the corresponding DNA segment from the other, identical chromosome. That segment detaches itself from the intact “sister” chromosome and moves over to the defective chromosome, helping to repair the damage. The gap created in the donor chromosome is refilled by using the segment from its remaining intact DNA strand (DNA consists of two matching strands) as a template. Both chromosomes end up with a complete, undamaged genetic segment.

In the new study, Prof. Zvi Livneh, head of the Biological Chemistry Department at the Weizmann Institute of Science, has for the first time observed this repair mechanism in action. Furthermore, Livneh and his team, which consisted of graduate students Ala Berdichevsky and Lior Izhar, also showed that the repair mechanism based on a genetic “donation” from the sister chromosome is unusually common: it is responsible for 85% of last-resort repairs – those performed by alternative repair systems when the major, “all-or-nothing” repair mechanism fails. The second last-resort system – the relatively inaccurate repair mechanism that allows the creation of mutations – is responsible only for some 15% of repairs.

Keep in mind the limitations of this repair technique. First of all, it doesn't work on males for X chromosomes since males have only one X chromosome. Also, the copied section may be different than what it replaces because there is considerable genetic variation between the chromosomes people get from each parent. Its possible that by doing the copy a harmful mutation that was silent on one chromososome could get copied to the other so that then two copies of the harmful mutation would exist in the same cell. Still, the vast bulk of the time when this repair mechanism is used the result is beneficial.

It is conceivable that some day this repair mechanism could be hijacked by gene therapy delivery mechanisms to replace sections of a chromosome with new and improved genetic code. The gene therapy could cause a piece of chromosome to be recognized as damaged, a mini-chromosome could be introduced that looked like the matching pair member so that the replacement DNA would be copied from the new mini-chromosome. So this latest research result may eventually be useful for genetically-based treatments.

By Randall Parker    2002 October 25 01:10 PM   Entry Permalink | Comments (2)
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