Genetic Engineering in Agriculture

Let’s talk about genetic engineering in agriculture. As we know, genetic engineering allows transferring genes from one organism to another. How is this useful in agriculture? We’re faced with a challenge over the next 50 years: feeding an ever expanding human population. According to UN estimates, human population will level off at about 10 billion people. Can genetic engineering in agriculture help?

A real problem in agriculture that existed for millennia is that most plants cannot grow in salty soils. When soil is irrigated, that is when people bring water to dry soils, the water also brings salts. This temporarily allows plants to grow, and normally these small amounts of salts get removed from the soil by rainfall. In dry climates, however, there isn’t much rain. As time goes on, salt builds up.

Salt build up has always been a major problem in agriculture. It led to the fall of civilizations. For example, the Mesopotamians fell as a civilization largely because of salt build up in their soil. Today, it is estimated that up to 65 thousand acres of farmland a day are lost to excess salt build up. The soils are essentially rendered unusable.

Salt is toxic to plants in two ways. First, salt impairs the roots from taking up water. Second, salt blocks several of the enzymes involved in important processes. How does it do that? It alters the way that these proteins fold, and if an enzyme folds incorrectly it won’t be able to do its function. The particular enzymes I’m talking about are involved in making proteins, and also some involved in photosynthesis. Photosynthesis is the process by which a plant converts solar energy into stored energy in the form of sugars.

Few plants in the world can thrive in very salty soils. Certainly, not the major crops (rice, wheat and corn). Finding a gene for salt tolerance in these crop plants is unlikely. If you go to the crop seed bank, it’s likely that you’re going to find a variety of rice that has a mutation that makes it tolerant to salt.

Scientists always use “model organisms” to do research. The model plant is a tiny mustard like plant, called Arabidopsis. Arabidopsis is a model for the genomes of the major crops. Arabidopsis does all the things that the major crops do. It has roots, stems, leaves, flowers and all those things. It is useful to study it because we can grow it in a greenhouse near a laboratory and we know its entire genome.

In the 1990’s, Eduardo Blumwald found that Arabidopsis has a gene that is expresses as a protein which suck ups salt form the soil, and put it into storage depots inside of cells called vacuoles. These particular cells are in the leaves of the plant. This might be a pretty good way to tolerate salt. The salt would never get into the rest of the cell.

The problem comes when the salt build up in the soil is very high, as happens in soils that had been rendered unsuitable for agriculture. There isn’t enough of this protein, so the excess salt leaks out of the vacuoles and gets into the rest of the cells.

Using genetic engineering, Blumwald has added a vector with a very active promoter (a section of DNA that turns on a gene) beside the gene that allows the salt to be stored in vacuoles. So, the expression of this gene would be enhanced. When he made transgenic plants using this vector, the genetically modified Arabidopsis was able not just to withstand salty soil, but to thrive in it. What an amazing thing!

Blumwald didn’t really want to grow Arabidopsis on salty soils, but to get this gene into crop plants. Genetic engineering allows transferring genes from one organism to another. When the active salt-tolerance gene from Arabidopsis was put into a tomato plant, it became very salt-tolerant. A normal tomato plant would wither and die in a salty soil, whereas the modified tomato plants would be just fine. What’s more, the salt was in the leaves, the tomato fruits were just fine.

While tomatoes are important, they are not nearly as important as the major grain crops. So, Blumwald and others are busily trying to transfer this salt-tolerance gene from Arabidopsis to rice, wheat and corn.

This may make salty soils in the world usable for farming. Salt-tolerant transgenic plants may make deserts bloom again.

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Ten Facts About the Human Genome

1. Of the 3.2 billion base pairs in the human genome, less than 2% code for proteins. 98% of the genome is not coding for proteins. There are 24000 genes in a human. Rice, for example, has 35000 genes. What is going on? I think I’m more complex than a rice plant.
   
   
2. 99% of the human genome is identical in all people.
   
   
3. The functions of many of the 24000 genes in the human genome are not yet known.
   
   
4. Genome sequencing is getting faster and cheaper. In 2007, the genomes of James Watson and Craig Venter were made public. It took those genomes two months each to get sequenced, with a cost of about 1 million dollars each. The price is going down and down. The objective is to reach the 1000$ genome by the next decade.
   
   
5. Genome sequencing of individuals is useful for: screening for possible diseases, genetic research and origins research.
   
   
6. We have fewer genes than a rice plant. There are three possible explanations for this, but the basic one is that we can do many things with each gene.
   
   
7. Genes are interrupted in their information sequence by non-coding sequences, called introns. Introns are kind of nonsense. Every gene has so many introns that the coding regions can be mixed and matched together. Each human gene gets translated into about 5 proteins.
   
   
8. After a protein is made, it folds into its three-dimensional shape, but other things can happen to it. Sugars can be put on to proteins and change their functions. We can have a set of genes that add sugars to various proteins. These modifications add to the diversity of proteins that can be made.
   
   
9. There is a big chunk of the human genome that makes RNA’s that don’t get out of the nucleus, that don’t end up getting translated into protein. These are very small RNA’s called micro RNA’s (mRNA). They were very recently discovered. In fact, they aren’t even in most textbooks. In the human genome, these small RNA’s may be involved in gene regulation (turning a gene on or off). We’re not quite sure what these mRNA’s are doing, but humans have a lot of them.
   
   
10. Scientists used to say that 98% of the human genome that doesn’t code for protein is junk. It looks like this junk has a valuable function. The junk does have some gems there, the mRNA’s.

The Genome Project History

What is the genome project history? Well, let’s review history. In the closing days of World War II, the United States exploded atomic bombs on the Japanese cities of Hiroshima and Nagasaki. Hundreds of thousands were killed. Many more were exposed to radiation. These survivors, and their descendants, had been intensively studied ever since that time for increases in mutations and their effects on people (especially cancers). By the late 1970’s, methods had been developed to get the sequence of DNA molecules. With this technology, scientists were able to study mutations and genetic diseases in much more detail. In 1984, Nobel laureate Renato Dulbecco made the daring suggestion that we should sequence the entire human genome. If we did that, we could compare the people who were not exposed to radiation to those who were, and we could get a very good handle on genetic damage. It’s not surprising that the first sponsor of what came to be known as the human genome project was the US department of energy, which oversaw the radiation damage project.

The human genome sequence was determined in two ways. There was a huge challenge: chromosomes are extremely long (on the average 100 million base pairs of DNA). Machines could sequence 800 base pairs at a time. So, we cut the 100 million base pairs into 800 bases fragments. How many fragments are those? Well, that’s your assignment. Hint: it’s a lot.

When we determine the sequence of each of those fragments, we just stick them together. Oh, yeah? How are we going to fit them together? How do you know which fragment is which? It’s like taking the Bible, cutting each word and putting them on the floor. How do you line them up?

There were two methods to get some kind of signposts by which we could say “this belongs here”. The methods were called hierarchical sequencing, and shotgun sequencing. Hierarchical sequencing was done by the government sponsored Human Genome Project. This was sponsored by many governments from around the world. Thousands of scientists worked on this effort, and it was led by Francis Collins.

They tried to recognize short sequences of DNA that were at certain landmarks all the way down the chromosome. If that short sequence is on the left end of the chromosome, for example, and then you randomly sequence fragments and you see that short sequence you’ll that belongs on the left end.

It took ten years for scientists to look at all the human chromosomes and get these landmark sequences. Then, they arranged them. It took about 15 years to do this human genome sequencing.

The other way to do it is called shotgun sequencing. Shotgun sequencing was done by private industry, led by Craig Venter. They did the same thing using with landmarks, but they had a computer to find them. This took amazingly sophisticated computer analysis. A whole new field was developed for this shotgun sequencing effort: bioinformatics. This was much more rapid, it took only a year to sequence the human genome.

The complete human genome was finally published in 2003.

Cancer Treatment

We’ve seen what cancer is and what we think causes it. What about cancer treatment? How do we treat and cure this fearsome disease? There are three ways to treat cancer: surgery, radiation and chemotherapy. Surgery is by far the most common cancer treatment. Removal of a tumor that is localized can cure cancer. Much of cancer surgery in recent years has become conservative of the surrounding tissue.

Radiation is used in cancer treatment on localized regions that can’t be removed in surgery. This might be in a very diffuse area, for example. It also might be useful to shrink a tumor that is up against a vital organ and the surgeon is afraid of damaging that organ. Radiation damages DNA, it causes both strands of DNA to be broken. The amounts of radiation used are small from the military viewpoint, but quite large from the medical one. Let’s illustrate this. The lifetime exposure to radiation of a typical person on Earth is about 0.12 gray (a physical unit). During the course of radiation treatment, the tumor itself gets 50 gray over five weeks. That’s 400 times the lifetime dose.

The third method of cancer treatment is called chemotherapy. It is used when tumors have spread over the body, because when you put a drug in the blood system, it will be distributed everywhere. Typical chemotherapy uses drugs that kill all dividing cells, including the tumor. There are side effects to chemotherapy. Normal tissues get affected, like bone marrow, cells in the intestines and skin.

A wide array of drugs that block cell division have been isolated and are used. Some are natural products we get from plants, others we build in the laboratory.

A Real Life Case

This is a real case involving cancer treatment with chemotherapy. John first noted that he tired easily at the gym. It got worst over several weeks. When he began to have shortness of breath even when he walked from room to room, he decided to see his physician. When his doctor looked at a drop of blood under the microscope, he saw many white blood cells. A blood sample was sent to laboratory, and they confirmed that he had over 200000 white blood cells per milliliter. This is 40 times normal. An hematologist looked at bone marrow as well as the blood and found a lot of immature white blood cells. In addition, he saw an abnormality: a funny looking chromosome called the “Philadelphia chromosome”. The diagnosis: chronic myelogenous leukemia.

No one knows how, but in this disease, the DNA in two chromosomes inside an immature white blood cell is cut and spliced. Two chromosomes exchange material, so that part of two genes that are ordinarily on separate chromosomes come to be right beside one another. The shuffled chromosome that was seen in John’s white blood cells was first noticed by scientists in Philadelphia in 1960, that’s where it got its name. White blood cells carrying this strange chromosome are stimulated to divide very rapidly. This is why they found this huge number of cells in John.

John was first treated with standard chemotherapy. He was given drugs designed to kill any reproducing cell. One of the drugs bound to DNA. A second one blocked the assembly of amino-acids. A third drug blocked the mechanism that partitions chromosomes to new cells. These drugs have bad side effects on other dividing cells. Using this conventional cancer treatment, John’s white blood cell count went down from 200000 per milliliter to 80000 per milliliter. This is still 16 times normal, however.

The drugs that John took blocked cell division all over the body. They were non-specific. John’s oncologist now tried a new approach: a specific drug. In the 1990’s, the molecular biology of this type of leukemia was described in detail. The new gene found in the Philadelphia chromosome was sequenced, and its protein product was studied. The protein turned out to be a terrific cell-division stimulant. It causes cells to divide without control.

Next, chemists at a drug company went into the laboratory and designed a brand new substance: a chemical that would specifically bind to and inactivate this new gene product in the tumor cells. At the University of Oregon, Dr. Brian Druker coordinated a clinical trial in which patients with chronic myelogenous leukemia were given this drug; to test for its safety and then its effectiveness. Patients like John, whose blood concentration of white blood cells was still high, were given the new drug and the result was spectacular. In John’s case, his white blood cell count went down to a normal 5400 per milliliter. He was cured.

The development of this drug, which is called Gleevec, is a great example of a new molecular approach to cancer treatment. The aim is to find out precisely what’s going wrong in a tumor cell and design rational treatments on this basis.

Precise molecular descriptions of the chemical biology of cancer are leading to new drug treatment for cancer and targeted chemotherapies.

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What Causes Cancer, Part II

This is a continuation on the last article on what causes cancer. In my last post we’ve seen how cancer is a multi-step disease, caused by a sequence of genetic changes, or mutations in certain genes. Here I want to look at the inherited or external factors that cause these mutations, and are thus what causes cancer. There are a couple of ways we can get these genes mutated or changed. One of them is to bring an active oncogene (a gene that helps a cell turn into a tumor cell) into a cell. If a cell has an oncogene that is not being expressed, a virus can actually bring it into the cell.

Cancer Caused By Viruses

Tumor viruses were discovered in a very interesting way, in 1910 by Peyton Rous. He got a call from chicken farmers in Long Island. The chickens were all getting these horrible tumors. So, he thought it might be an infectious disease. He went to Long Island and he showed that there was a virus that was spreading form chicken to chicken and causing these tumors (sarcoma). He isolated the virus and then infected an animal that didn’t have the tumor. The animal got the tumor, and from the tumor in that animal, he isolated the virus and repeated.

Rous was awarded the Nobel Prize in 1966, 56 years later. Why did it take that long to recognize him? Because no one really wanted to believe that cancer could be a communicable disease. People started to look at proteins and DNA, and attention shifted.

Tumor viruses, as it turns out, have actively expressed oncogenes. There is a tumor virus that has an actively expressed oncogene for that growth factor. When these active oncogenes get unto our cells, they stimulate cell division. Approximately 10% of human cancer is caused by viruses. In our society there are two major ones that are important: hepatitis B virus is associated with liver cancer, and a papillomavirus (warts), which causes cervical cancer. In both cases the good news is that antiviral vaccines are being used and developed.

Inherited Cancers

Inherited mutations in tumor suppressor genes can allow cells to divide inappropriately. It turns out that about 10% of cancers are inherited. This includes about 10% of breast and colon cancer.

Compared to non-inherited cancers (called also sporadic), inherited tumors typically occur earlier in life. For instance, inherited breast cancer would manifest in women during their 20’s, instead of in their 50’s, as it is common with sporadic cancers. These inherited tumors also appear in numerous places.

After looking at the data, Arthur Knudson proposed the “two-hit” hypothesis. From Mendelian genetics we know that most cells have two copies of every gene. Knudson proposed that for tumor-suppressor genes both copies have to be mutated. Both alleles of the tumor-suppressor gene must be off. People with inherited cancer have already in all of their cells one bad allele. So, if they later get a mutation of the second allele, they’ll get a tumor. That can happen reasonably soon. People who inherited two normal alleles, however, need two mutations, and that’s going to happen somewhat later.

Cancers Caused by External Factors

Most cancers turn out to be sporadic, so they need many mutations in order for the tumor to form. What do I mean by many mutations? Mutations must happen not only on the tumor-suppressor genes, but also on oncogenes and metastasis genes. You need all these events to occur in a sequence.

A doctor at John Hopkins University, Bert Vogelstein, and his colleagues, spent a lot of time during the 1990’s looking at the sequence of genetic changes that lead to cancer, specifically to colorectal cancer. This cancer develops first as a small group of cells. This group gets larger and larger over time. You can actually follow this. In colonoscopy, the objective is to remove the group of cells before it becomes a tumor. If you remove the cells, you have a sample of an early cancer.

What Vogelstein did was to make a survey of all the genes involved in cancer in these groups of cells as they went through the stages of development. What he found was a very dramatic series of events. First there were mutations in tumor-suppressor genes, and then there were mutations on oncogenes. Then there were mutations in metastasis genes.

Describing cancer in this amount of molecular detail is an amazing achievement of modern biology and medicine. It would lead to treatments.

So, when we are talking about something causing cancer, we mean that they cause these genetic changes. DNA can change in two ways. First, there are spontaneous mutations that occur during DNA replication. This means that people can get cancer without getting exposed to any bad stuff. This is rare event, but think of the fact that the body has trillions of cells, and cancer can come from one cell that goes bad.

The second type of mutations is induced ones. They are caused by something from the outside. Most cancer mutations are caused by the environment. Cigarette smoke, for example. The chemical compounds present in cigarette smoke damage specific tumor suppressor genes. Ultraviolet light from the sun causes skin cancer.

However, these things are obvious. For the case of most cancers, we really don’t know what’s going on. In fact, many carcinogens are natural substances in the diet. In most cases, we just haven’t identified them yet. They’re not the things you might think of. They’re natural molecules that plants make to defend themselves. How do we know this? Because there are country differences in cancer incidence. Also, when someone moves from one country to another, they and their offspring will adopt the cancer rate of their new country. When you look very carefully at it, you find that it’s probably diet.

This is probably why cancer is so difficult to treat or prevent: in most cases we don’t even know what causes it.

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What Causes Cancer

What causes cancer? We all know that cancer cells are different from normal cells. One of the hallmarks of cancer cells is that they lose control over cell reproduction. Normally, cells control when and where they reproduce. They do it in two ways. There are internal controls. Normally, there is an internal clock inside a cell that triggers in the cell division cycle.

Most mature(specialized) cells do not divide. Immature cells divide rapidly (think of an embryo or bone marrow). What keeps cells dividing is a group of proteins that stimulate them. What keeps them from dividing is a set of proteins that blocks them from cell division. Because they block tumors from forming, we call them tumor suppressors. These proteins act the control points during cell division.

Cancer cells have defective tumor suppressors. They have mutations in their DNA that causes the protein that normally functions to block cell division to not function. Those are the internal controls of cell division.

There are also external controls in cell division. The external controls are hormone-like substances called growth factors. They stimulate cells to divide by acting like a gas pedal in a car. For example, a blood clot forms and then the wound heals, so the skin around the blood clot heals, and the clot goes away. The cells that surround the blood clot have to be stimulated to do so. Those cells have to surround the blood clot and close the wound. “Closing the wound” is not a biological term, what really happens is a lot of cell division. In turns out that the same cells that form the clot stimulate the cells above it to heal. The clot makes a growth factor that stimulates the skin cells to divide, reproduce and heal the wound.

Genes whose protein products stimulate cell division in this way are called oncogene. Cancer cells usually make their own growth factor. They would have an oncogene that is mutated, so it is producing constant stimulation for cell division. Other cancer cells may have genetic changes that make them hypersensitive to even tiny amounts of growth factors. These are the genes that turn on the gas pedal of cell reproduction.

The most fearsome aspect of cancer is that cancer cells can spread to other organs. This is called metastasis. You can’t do surgery if a tumor is all over the place. This leads to multiple organ failures, and it is really what often causes people to die of cancer.

Normal cells have a “glue” that causes the cells to stick to one another. This glue is specific. If you pinch your skin, you will notice that the cells at the skin stick together and are separate from the bone. They must have a specific “glue”, which is formed by proteins. There are cell recognition and adhesion proteins. Cancer cells loose this adhesion.

There are several steps in metastasis. First, cancer cells have to loose their adhesion and detach from the tumor. Then, the cells make it to the bloodstream or lymph system. In the blood, they travel to an organ. They stop at the new organ and grow as a satellite tumor. We know all of these steps in great detail at this point.

This events: inactivation of tumor suppressors, activation of oncogenes and metastasis; occur in sequence. Cancer is a multi-step disease. To be continued…

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Benefits of Genetic Engineering

What are the benefits of genetic engineering? The benefits of genetic engineering are the products we can make with it. Genetic engineering could be used to make products in two ways: we could take the existing organisms and modify them genetically to make them more efficient. We could for example add a promoter that would make the organism produce more of one kind of protein. The second use of genetic engineering is that we can take new genes and insert them into organisms that never had them before.

The first major product of genetic engineering was human insulin. Insulin is a protein that acts as a hormone to stimulate uptake of blood sugar into tissues, such as the liver and the muscles. In diabetes, people don’t make insulin. Previously, insulin came from slaughtered animals. The insulin protein has 51 amino-acids. The insulin from slaughtered animals is similar to human insulin, but very often is one or two amino-acids different. It still has the same function, but the human immune system will recognize these one or two differences. So, in a significant number of diabetics, when taking non-human insulin, their immune system reacts against it.

Here, what we need is human insulin. Insulin is made in very small amounts. So, the only way to get this in reasonable amounts is by amplifying the expression of genes for insulin in recombinant DNA.

At City of Hope Medical Center in California, Keiichi Itakura went into the chemistry lab and made the insulin gene. It wasn’t even then a big deal to make it. Itakura’s colleague, Art Riggs, took this gene and put it into an expression vector, right next to a promoter. The expression vector now had the gene and a marker. The expression vector was then put into bacteria, and the bacteria expressed human insulin. This is about 7 or 8 years after the first emergence of recombinant DNA technology.

The insulin then was extracted, sent to a drug company, and then to physicians. This is the source of all insulin now used to treat type 1 diabetics. This is really the great example of a genetically engineered medication.

Another example is the blood-clotting protein that is missing in hemophilia. This blood-clotting protein can be supplied with genetic engineering. People no longer die of hemophilia thanks to genetic engineering. So, here we have two clear benefits of genetic engineering.

Proteins used to treat diseases

There is a protein called Erythropoietin (EPO). EPO is a hormone-like substance made by the kidneys. It enters the bloodstream, goes to the bone marrow and stimulates the production of red blood cells. Red blood cells only last about 120 days, so they must be constantly replaced. There are many people with kidney failure for various reasons. These people are treated with kidney dialysis, because kidney transplants are limited.

The kidney filters out the poisons and keeps the good things. In this case, all you can do is to filter the bad things with the dialysis machine. Among the things that get filtered out is EPO. So, these people with kidney failure have kidney function restored by dialysis, but they’re not making EPO. They have severe anemia. The only way to get around this is by massive transfusion or to treat them with EPO.

The gene coding for EPO was isolated, EPO was made by recombinant DNA technology, and this is now widely used for people who are undergoing kidney dialysis and also people who are being treated with cancer chemotherapy. Many of the drugs used in chemotherapy destroy bone marrow cells.

EPO is also the first genetically engineered drug of abuse. Athletes found that if they take some EPO they can increase their blood cell count. Increasing the amount of red blood cells by about 10% can give an athlete an edge in competition. There had been great controversies in cycling and elsewhere because of the abuse of EPO.

In Animals

Plants and animals can be genetically engineered to make products useful for us. The great example of this is diary animals. Sheep, goats and cows produce a lot of milk. Biologists found that the expression of genes for the major milk proteins is under the control of a promoter. This promoter is a sequence of DNA that causes the adjacent genes to be expressed in the mammary gland. It is called the lactoglobulin promoter.

This sets up a really nice opportunity for using genetic engineering. You could take the gene you want expressed in milk and put it into a DNA vector. Then you put this vector into a sheep egg cell. If you do this, the egg can then be developed in the laboratory for a couple of days until it becomes an embryo. You can insert the embryo into a mother and the offspring that are born are sheep that would make milk which contains this extra protein. This was actually behind the reason for cloning Dolly the sheep.

There are a significant number of humans that lack adequate amounts of growth hormone. These people are very short in stature. The growth hormone is a protein. So, we can get that protein from the body, but it is made in the pituitary gland in extremely small amounts. So, again, we got to get it through genetic engineering. The gene was isolated, put into a vector, put into cows, and there is a herd in Argentina of ten cows that in their milk will supply the world need for human growth hormone every year. This process is called pharming, and a very promising technology.

In Plants

Plants can be genetically engineered to make useful products. Genetically engineering a plant is a lot easier than animals. We don’t need to inject and expression vector into the fertilized egg of a plant, because plant cells are totipotent. We can take any plant cell grown in a laboratory, put the vector in, and then grow the plant up from that cell.

Plants produce a lot of protein. For example, tobacco plants have been genetically modified to make TPA in their leaves. Tobacco leaves are very large and it is easy to get the TPA from them.

You’ve probably heard the term antibody. Antibodies are what the immune system makes to fight diseases. A “plantibody” is a plant is making a human antibody. We can use genetic engineering to have a plant make a vaccine. For example, the vaccine against bacterial meningitis has been expressed in the fruit of plant, a banana plant. The people still have to eat the fruit, but it doesn’t have to be refrigerated and you don’t need health professionals to administer it.

We can create plants with new capabilities. For example, a major component of detergents is lauric acid. This molecule is made in a biochemical pathway in tropical plants, such as coconuts and palm trees. A major source of it is palm kernel oil. Scientists pinpointed one of the key enzymes that is in the biochemical pathway for making lauric acid. Scientists pinpointed the gene in palm trees, they cloned the gene into an expression vector and put it vector into rapeseed plant that produces canola. Normal canola produces oil that is 0% lauric acid. This particular transgenic canola makes 60% lauric acid.

There is a viral disease caused by the tobacco mosaic virus. This is a major pest of tobacco. It doesn’t kill the plant, it kinds of destroys leaf tissues and spreads very rapidly. This virus reproduces in the cells but doesn’t kill the entire plant. The viral genome can be replaced with other genes; in this case, with genes for vaccines. Now, instead of making a lot of proteins in viral particles, these tobacco plants are making large amounts of a vaccine. This may be a new use for what is widely grown crop around the world.

These are just a few benefits of genetic engineering. I’m sure more are coming in the future, as we discover more and more about genes and proteins.

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Advantage of Stem Cell Research

What is the advantage of stem cell research? There is a need for new cells in medicine to replace cells that are damaged. For example, in a heart attack, the heart muscle is damaged, and it is usually permanent damage. How are you going to replace that tissue? In the brain, Parkinson’s disease and others result from a lack of functional cells. Diabetes, specially type 1, the pancreas is damaged. Let’s get new cells to replace these! The problem is where we’re going to get them. Well, you could do a heart transplant, or other organ transplant. Organ transplants are hard to get. There are many more people waiting for kidney transplant than we have kidneys available. So, here is obvious the advantage of stem cell research.

The second problem is that the immune system ultimately will reject the transplant. A person who gets an organ transplant must take immune-suppressor drugs to keep the organ as long as possible.

What about using stem cells? Stem cell transplants are already performed every day. Bone marrow gets damaged when cancer is treated with radiation therapy and chemotherapy. All the cells, including the stem cells inside the bone marrow, are damaged. A person who is treated with radiation and chemotherapy for cancer is going to be severely anemic and immune-compromised; because their immune system would not be working (white blood cells would not be produced in sufficient numbers).

The strategy is the following: if the patient’s bone marrow is removed before therapy and stored literally in a refrigerator, “reinserted” after therapy, it has enough stem cells to form the new blood cells. These stem cells are called pluripotent. They are not totipotent (like the ones found in an embryo). They can give rise to all of the blood cells, but not others.

What about a patient who has blood cancer. The patient’s bone marrow can’t be used. You need a genetically matched donor to give his stem cells. You’ve probably seen advertisements in your community for becoming a member of a bone marrow registry. It is not a hard test and it is a wonderful thing. Imagine it; you could save a life with a very simple procedure of donating some stem cells.

Stem Cells From Your Own Fat

As a plastic surgeon in Los Angeles, Dr. Marc Hedrick’s practice included liposuction, where unwanted fat is removed from the body. Instead of throwing it away, he asked if there was something useful in it. When he looked at the fat under the microscope, he saw not just fat cells, but some other cell types as well, including what appeared to be bone and cartilage. Hedrick proposed that these specialized cells (bone and cartilage) got there because fat tissue must have some stem cells.

Hedrick knew about stem cells in bone marrow from his medical training. These are the stem cells that constantly replenish the population of red and white blood cells. When he took them and implanted them in animals, they would specialize into the tissue where they are located. In contrast, if you take a specialized cell and put it into another environment, it stays the way it was.

So, stem cells from fat can be put into damaged blood vessels and specialize into blood vessel cells, thus repairing the damage. Fat stem cells are reaching the clinic. The advantage of using fat stem cells is that a person’s own fat can be used to get them. They won’t be rejected by the immune system.

Hedrick and his colleagues have invented a way to get fat stem cells in about an hour in the operating room, while the patient is there waiting for the implant of stem cells. He needs about a pound of fat. That’s enough to get 200 million stem cells. This is enough for therapy. Recently some women in Japan received fat stem cells to help repair breast tissue after surgery for breast cancer. In Germany, a child got his own fat stem cells to help repair his skull after damage in an accident.

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What are Stem Cells

What are stem cells? Stem cells are unspecialized cells in the body that constantly divide to form a pool of cells that can then specialize when they are needed. When we have the union of the sperm and the egg to produce one cell, this in turn divides into a form called morula. The morula contains identical cells all of which are totipotent. This means that any single cell in the morula can divide and become virtually any cell in the human body, including embryonic tissue and placenta. The big change comes when it divides.

In the blastocyst, the cells become more specialized, but they still retain some potential to differentiate into almost any of the cells of the body, except some specialized ones like the placenta.

What we’re interested in is in what happens from here on. With each division, cells can differentiate from its parents and specialize more and more. They loose a little bit of their power to become anything. They go from pluripotent to multipotent. Multipotent cells are specialized cells which nonetheless have the ability to become a variety of cells. The multipotent hematopoietic cell, for example, which is the cell that forms blood elements, can become very varied elements in the blood.

These multipotent cells have the ability of maintaining an undifferentiated state.

Look at the picture. If a stem cell undergoes mitosis, it divides and produces two daughter cells. One of the daughter cells is exactly like the original stem cell. This one maintains the pool of stem cells from which to get new cells all throughout our lives. The other cell goes on to differentiate, going different pathways to become anything except a placenta or other early embryonic tissues.

When a cell goes all the way through a pathway, it can’t go back. This means that it cannot divide again and become less differentiated.

The ultimate stem cells are embryonic stem cells. They are totipotent. They can become any cell in the organism. At about the tenth day stage after fertilization there are several dozens of these undifferentiated totipotent cells in a human embryo. These cells can be removed from the embryo, grown in a laboratory dish and reproduced. In 1998, James Thomson at the University of Wisconsin showed that this is possible to do. Put them in a laboratory dish and they would grow indefinitely as a laboratory culture.

In laboratory experiments on animals, these embryonic stem cells can be induced to form many different cell types. In animals, these cell types coming from embryonic stem cells have cured brain damage, heart damage, muscle damage, etc. This has generated great excitement for their potential in human medicine.

The proposal is to use laboratory grown stem cells as a supply. You don’t need a lot of embryos to do this. The problem is that if I get some stem cells from someone else, they’re not mine. Those cells going into my heart would do the work, but then my immune system would ultimately reject them.

This has led to the proposal of therapeutic cloning. This is the subject of another article.

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Pros and Cons of Cloning

What are the pros and cons of cloning? Human clones are born every day: identical twins. An identical twin is the product of an egg that divided into two, and each one of those formed an embryo. They’re genetically identical. Since cloning has been done on a number of animals, it is possible to do deliberately do human cloning. Why would anyone want to do this? For a couple of reasons. These are some of the pros of cloning:

- Cloning might be useful for people who have problems with normal reproductive mechanisms. For example, a woman might not be able to make eggs but want a child related to her. She could be a donor to make a clone carried by another woman. This is just another reproductive technology, like in vitro fertilization. You may recall that in vitro fertilization was greeted with horror and now is more or less a routine procedure.

- There might be a desire to perpetuate valuable genotypes. You might have an old woman who never gets cancer. She might be carrying certain mutations and you want to study them. It might be better to study them if the person is cloned

- We could clone a unique individual, like an Einstein, to study the genes that made this person unique.

- Perpetuation of a dying child. There had been movies made of this possibility, where the child is dying and the parents want the child back.

There are several concerns about doing human cloning. These are the cons of cloning:

- The process is not a very efficient one. Dolly was one of 277 tries, all the other failed. Things have improved somewhat, but it isn’t as good as in vitro fertilization.

- In some species, clones have medical problems. Dolly the sheep died young. Scientists claimed that Dolly did not die of anything to do with cloning, but other animals have died young, there have been defects in the immune system and disease susceptibility in clones.

- There are a lot of unknowns. No scientific group is seriously proposing human cloning.

Some Other Advantages of Cloning

There have been proposals to use stem cells to regenerate organs and tissues. The problem with this process is that if I get stem cells from someone else, they’re not mine. Those cells going into my heart, for example, will be rejected by my immune system, just like any transplant organ.

This has led to the proposal of therapeutic cloning. Here’s the case: I have a damaged heart because of a heart attack. My skins are removed and grown for a few days in the laboratory, then sent to a cloning center. In the cloning laboratory, a woman has donated an egg cell. Her egg cell nucleus is removed and replaced by my skin cell nucleus. The egg is then stimulated to divide and forms an early embryo. This is actually possible to do in human medicine today.

After ten days, the embryonic stem cells of this embryo, which are genetically mine, are removed, placed in a laboratory dish, and induced to form heart cells. These heart cells are then implanted in my heart, where they repair it.

So, these are some of the pros and cons of cloning. I particularly I’m pro therapeutic cloning, although, new methods are been developed that are more efficient than cloning, specifically in genetic engineering. I would talk about them in another post.

Who Invented DNA Fingerprinting

Who invented DNA Fingerprinting? In the early 1980’s, Alec Jeffreys developed DNA fingerprinting at the University of Leicester. He was studying genetic differences between individuals of various species. He was comparing the genes of seals and humans, and to his surprise, he noticed common short repeated sequences. When he looked at the human ones, he found these short repeats that were similar between parents and children. He also noted that the children’s sequences were composite of the parents. This meant that they were inherited. Jeffreys published his findings in 1985, and the genetic floodgates opened.

Read this pages to learn more about DNA fingerprinting:

• How Does DNA Fingerprinting Work
• DNA Fingerprinting Uses

DNA Fingerprinting Uses

What are some DNA fingerprinting uses? We’ve seen how DNA fingerprinting works, but what is it good for? There are a number of interesting examples of DNA fingerprinting uses. Alec Jeffreys invented DNA fingerprinting in 1985, and his method immediately was used to solve some serious issues. The first case that came to his attention was involving immigration. A family from Ghana had immigrated to the United Kingdom. When one of the four sons of the parents went for a visit to Ghana, he tried to come back and was detained at the airport by British immigration authorities, because his passport just wasn’t right. The authorities claimed that he wasn’t the son at all, but a cousin from Ghana who was trying to sneak into the country.

Jeffreys did DNA analysis of the mother and the three undisputed sons, as well as the son in dispute. The result was that he was definitely the son, and they allowed him to the country. Here we have an interesting DNA fingerprinting use, solving an immigration issue.

Criminal Cases

Soon, Jeffreys was called to a criminal case in Leicester. Two girls had been raped and murdered in the same area under similar circumstances two years apart. A man in jail confessed the second crime, but claimed innocence of the first one. Jeffreys did DNA fingerprinting of the victims, the suspect and semen that was found on the victims. As a result, Jeffreys concluded the same man had committed both crimes. It wasn’t the man who confessed, though. Why this guy confessed no one knows.

The police then asked all men in the area to give a blood sample. Five thousand men did so. When DNA analysis was done, there was still no match. Coincidentally, a woman overheard a man saying: “I gave two blood samples, one for me and one for a friend who didn’t want to give it”. The police found the man and asked him to give a sample. When they did the analysis, he turned out to be the killer.

DNA fingerprinting, as a result of this case, is now widely used in forensic cases.

Identifying People

Abhilasha Jeyarajah was a baby who was torn from his mother’s arms when, in 2004, a Tsunami hit Sri Lanka. Amazingly, Abhilasha survived. While his parents franticly looked for him, the baby was picked up by a local teacher and brought to the regional hospital. It was the worst day imaginable for the hospital staff. Hundreds of dead and dying children and adults were everywhere. When Abhilasha was brought in, the nurses were surprised to see that he was alive and healthy. It was a true miracle. He quickly became a celebrity in the hospital. Because he was the 81st infant brought in that day, they called him “baby 81”.

A few days later, his frantic parents came to the hospital, where they heard there were unclaimed babies. Joyfully, they were reunited with Abhilasha. “Not so fast”, said the hospital staff. In the previous two days, other couples had come to the hospital searching for their missing babies. Eight couples had claimed baby 81 was theirs. The question ended up with a judge.

There is a story in the Bible where King Solomon faced a dilemma of ruling which of two women was the true mother of an infant. Wise Solomon said: “I’m going to cut the infant in two so that each of you can have a half”. Of course the real mother was immediately revealed. In Sri Lanka, the judge had a worst case. He had nine couples claiming to be the parents of a six-month old boy.

Unlike Solomon, who just had wisdom, the judge had DNA for identification. Testing by molecular biologists soon found the real parents.

DNA in History

In 1918, with the Russian Communist Revolution raging; Czar Nicholas II, his wife and three of their children were killed in a town and buried in a shallow unmarked grave. In 1991, in a not Communist Russia, two amateur historians found what they thought was the grave of Nicholas II. There were two older people and three younger people. The sizes of the skeletons were consistent with the family. There were golden dental fillings. At that time not everyone in Russia could afford gold fillings.

The skeletons were too damaged for further identification, but fortunately, the bones had DNA. The short tandem repeats where compared in the bone with those of survivors of their family. There are surviving members of the Romanov family (the emperor’s family). For example, a great granddaughter of the Czar’s sister is still alive. The great grandson of the Czar’s aunt is still alive. Also, the body of the Czar’s brother was exhumed and some DNA was there.

The result: the same repeats were present in the dead family. That proved that they were from the Romanov family. There was a huge military funeral with full honors.

DNA fingerprinting has been used in many more instances. It was used to identify victims in the World Trade Center terrorist attacks in the United States in 2001. Many of the victims were beyond identification by any other means.

DNA databases are being built all around the world. For example, for some time in the United Kingdom, every one arrested for serious crimes has been DNA typed. There are now 3.5 million people in their database. In the United States there had been a number of propositions dealing with DNA typing for arrested people. The FBI has 3 million DNA stored and typed.

How Does DNA Fingerprinting Work

How does DNA fingerprinting works? DNA fingerprinting is a reliable method for identifying people. How DNA fingerprinting works as to do with something called the HLA system (human leukocyte antigen system). The HLA is a system that codes for proteins on the surfaces of many cells, including white blood cells. HLA genotyping is used in transplants. With this system, people could match transplant genetically, so that a transplant from one person to another wouldn’t be rejected by the recipient.

There are four different HLA genes, labeled nicely A, B, C and D. HLA A has 23 alleles. HLA B has 47. HLA C has 8 and D has 23. A person could be, for example, A11, B16, C5 and D11. With more alleles is more likely that people would be different from one another, and that parents and children can be matched better.

There are a couple of problems with the HLA system, however. First, you need well preserved tissue or blood. That’s a little difficult sometimes. Second, HLA proteins are not always present in our cells. Third, a lot of mixtures of these genes go on when gametes are produced. Fourth, there are pretty common HLA alleles, and some extremely rare ones.

The Short Tandem Repeats

Human genome sequencing has revealed that the genome contains short sequences that are repeated many times in tandem. These are appropriately called Short Tandem Repeats (STR). For example, let’s consider the DNA sequence TCAT. Looking through the whole genome, there are different Short Tandem Repeats, and the repeat numbers are inherited. You might inherit one chromosome that has TCAT repeated five times, and the chromosome from the other parent might have TCAT repeated seven times.

You might ask how this block repeat happens. Molecular biologists, believe it or not, say that it is not clear how it happens. They have some ideas, though, but it is not clear. There are 10000 of STR’s scattered throughout the genome, but for DNA fingerprinting purposes, we use typically 13 of them.

These 13 repeated sequences are polymorphic. This means that there is more than one type of repeats. I might inherit five repeats of TCAT at a certain location, and seven from my other parent. If we were all the same for this repeat number, they wouldn’t do any good. These different numbers of repeats are what set us apart.

To analyze DNA this way, however, we need first to make a population survey. We need to know the frequencies of the alleles. For example, five TCAT are present 50% of the time in the population, and seven is present 50% also. If somebody comes across and has ten would be a totally different individual.

Supposing we are dealing with two of these 13 short tandem repeat chains, and that they have three alleles: A, B and C. Let’s say that the A allele is frequent in one person in a hundred. The B allele is one in five. The C allele is the more common, four in five. For the second short tandem repeat: A allele one in ten, B one in two, C two in five. A, B and C are just the number of repeats. A for example might be five, and B seven repeats.

So, let’s review:

Short Tandem Repeat 1:
A: one in a hundred
B: one in five
C: four in five

Short Tandem Repeat 2:

A: one in ten
B: one in two
C: two in five

Here is the key argument for doing DNA identification in this way. It comes from Mendel and probability. For a person to be carrying both the A and B alleles of STR number 1, the combined probability is the product of the two probabilities. The combined probability of A(1/100) and B(1/5) is:
The probability for STR number 2 A(one in ten) and B(one in two) is:

So, what is the probability of having both of them at once? Yes! It is the product of their probabilities:

We’re now getting a pretty low probability. One in ten thousand! This is the probability of carrying the four alleles: A and B of STR 1, A and B of STR 2. Think about it. We have 13 different systems for identifying people. If we’ve got these different alleles, the probability of two people having identical alleles is vanishingly low. It turns out that we’re all virtually unique in these sequences. That’s why DNA fingerprinting is so useful in identifying people.

What is Genetic Engineering

What is Genetic Engineering? Genetic Engineering is the manipulation of microbes, plants and animals to make products that are useful to people. As such, genetic engineering is not new. It began a long time ago. I would say that genetic engineering, or biotechnology, began with agriculture. Agriculture I define as the harvesting, planting of cultivation of plants for food and fiber. This was the first biotechnology. Estimates are that agriculture probably began about 10000 years ago, in what is now the region near Iraq.

We have evidence that Sumerians living there at the time learned that barley plants growing around their homes made seeds that could be used to make bear and bread. They started growing these seeds near their settlements. They would use some of the seeds to make bear and bread, and then they would grow the rest of the seeds nearby.

In ancient Egypt, the hieroglyphic symbol for food is a picture of bear and bread. You may ask, why bear? Just to have a good time? Answer is no. When people began to live in settled places, they realized by trial and error that water purity was a big issue. As they would be going to the bathroom in the same water they were drinking, people would become sick. Alcoholic beverages kill most of the bad things that are in water. It’s not surprising that they were invented quite early on by humans.

The process of making alcohol from seeds is actually carried by yeast cells that live in the grain or grapes. This process is called fermentation, and it is a form of biotechnology. Modern genetic engineering, however, refers to the use of a technique called recombinant DNA. To illustrate what is genetic engineering and what are its uses I’d like to tell a story:

As Tom drove home from work, he felt his face twitching and had a really bad headache. Tom was one of the several million people who have a stroke each year in the United States. A blood clot was blocking an artery leading from the heart to his brain. This deprived brain cells of oxygen, and irreversible damage to the brain could occur quite quickly. When you think of blood clots, you got to consider that they do go away eventually. “Go away” is not a really good biochemical term. We say that the clot dissolves, but this happens rather slowly. The way this happens is the following: as the wound is healed, a series of cells that is healing the wound make a substance called TPA (Tissue plasminogen activator), which activates the blood clotting system. The clot then dissolves.

The time factor is important biologically speaking. If the clot dissolved the minute it was formed it wouldn’t do much good. The blood would flow out and you’ll lose it all. Slow dissolving of the clot is a good thing, but not for Tom. Tom was having a stroke, and that’s not a situation in which we want a blood clot for a long time. Every minute the blood flow to the brain is blocked is harmful.

Luckily for Tom, he was near a hospital. He drove to the parking lot; the emergency stuff got him and immediately injected a drug right on the surface of the blood clot. The clot dissolved right away, blood flow was restored and there was minimum damage to the brain. Tom was home the next day and he was fine.

The drug that the emergency room stuff had injected was TPA. This drug is also called PLAT in medical terms. This is the protein that initiates the clot-dissolving process. Without using TPA, the clot would have gone away, but his brain would have gotten damage in the meantime. Adding this substance right away to the site of the clot activated the clot-dissolving system right there.

TPA is made in very small amounts and only when it is needed. The bad thing is that if we want to use that as a medication, we’d better get a lot of it. It’s virtually impossible to get enough TPA from the cells of a body to store in the emergency room. This is when genetic engineering enters the scene.

First, DNA was extracted from human cells. The gene coding for TPA was isolated and inserted in hamsters. This allowed to produce TPA in amounts far greater than you could ever extract from blood. The TPA is then purified and sat on the shelves ready for a patient like Tom.

The scenario of using a gene to produce a useful protein, by this genetic engineering technology, has now been played out for dozen of products. This is part of a revolution. The revolution of genetic engineering.

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