Targeted Gene Replacement



Contained within every cell of our bodies is a set of instructions, a veritable book if you will, that specifies its function. Not every page of this book is being read by the cell at the same time, because different cells have different functions. For example, epithelial cells don't fire off action potentials as muscle cells do, or relay chemical signals as neurons do.

This book has within itself enough information to allow a single-celled embryo to become a newborn child; and this book of instructions is different for each one of us.

This book filled with pages of information, in case you haven't already guessed, is the genome. The genome is written in the language of nucleotides, a language that contains only four letters. A standing for adenine, C standing for cytosine, G standing for guanine, and T standing for thymine. The different sequences of these letters make up words we call genes, which convey information and are the functional units of the genome. In humans and in mice, this genome contains 3 billion nucleotides. To give you a sense of how much information that is, if the genome really were a book, it would constitute 1000 volumes, each with a 1000 pages.

Over the last several years, scientists have developed techniques for specifically changing a paragraph, a sentence, or just a single letter of the genome of a mouse In effect, they are rewriting portions of the book, which will enable them to see the affect these altered instructions have on the developmental functioning of the mouse.

Introducing specific changes into the letter sequence of a gene is called gene targeting, and is being largely done on mice. The reason for this is that the genome of a mouse is approximately 99% homologous with that of a human. Thus, the hope is that what is learned from these experiments on mice can be applied to humans.

These types of experiments done on mice have already shed light on the complex process of human embryonic development and immune system response. There are hopes that gene targeting will also help in explaining the functioning of the human brain and how diseases arise from defects in human genes. Those involved with the genome project also have taken a keen interest in gene targeting, since it will hopefully expand on the knowledge generated by this undertaking.



Contrary to what many people think, genes are not actually for any one trait. Rather the nucleotide sequence of a gene specifies the amino acids that need to be looped together to make a certain protein. It is in fact proteins that carry out most of our cellular activities. Proteins may function as enzymes, structural components of the cell, or as signaling molecules. Since a sequence of nucleotides alone is not enough to clue us in to all of the minute functions of a protein, gene targeting is used to take our investigations to a deeper level.

Gene targeting has its pros and cons. One could say that we are once again playing God and are making descions about what is normal and what is not. On the other hand, gene targeting provides us with a way to see how genes control various biological processes. Gene targeting is also very useful in dealing with complex organisms such as mammals, where classic methods of genetics fail.

For example, if geneticists want to determine how a single-celled organism such as yeast replicates its DNA, they expose a large number of individuals to a chemical that will damage their DNA, otherwise known as a mutagen. This process will ensure that each individual carries a mutagen in at least one gene. From here, geneticists can readily identify those individuals not capable of replicating their DNA. Scientists use large quantities of bacteria or yeast when doing these experiments in order to ensure that mutations in each of the genes responsible for DNA replication show up. And once the genes that play a role in DNA replication have been identified, scientists can determine their specific role in the replicating process.

When it comes to multicellular organisms however, things get much more complex, as the size of the genome increases. For example, Drosophila Melangaster, and a small worm called Caenorhabditis Elegans are multicellular organisms often used by geneticists in gene targeting experiments. The size of the genome increases greatly. While a single-celled bacteria has 3,000 genes, that number jumps to 20,000 with the common fruit fly, and the genome of a mouse is ten times that number. The more genes, the more complex their interactions become. Following the effects of any one gene thus becomes a huge undertaking.

Size of the organisms being studied also plays a role, for while it is relatively easy to study a billion or more bacteria at one time, that number becomes incredibly impractical when you are dealing with mice or even fruit flies. A reasonable amount of mice to study in a gene targeting experiment would top out at around 1000 specimens.

To make things even more difficult, most multicelluar organisms such as mice and drosophila are diploid. Thus, in order to see signs of a defect or abnormality, both of the organisms copies of the gene must be damaged. Scientists can create such individuals by mating parents who each have a mutation in one copy of the relevant gene. As we all know, 25% of the offspring will thus be homozygous recessive and show the defect. However, this takes time and many mutations scientists are interested in are often underrepresented.

Gene targeting gets around these difficulties I have just described, because investigators are choosing which gene to alter, and they thus have almost complete control over how the gene is modified. Gene targeting allows science to determine how intricate biological processes occur.

The mouse is a favorite choice of geneticists to study, because as mentioned before it is quite similar to a human in terms of genome size and function and it is small and easily reproduced. Again, if we were dealing with classic methods of genetics and simply studying mutations that already existed in the population, many abnormalities would be underrepresented, because mice with certain more severe defects would be spontaneously aborted. This is why gene targeting is so effective, since scientists can in a sense "tailor make" mutations to suit their studies.

In initial experiments, the chosen gene is often completely knocked out of the organism in order to see the consequences it would face without any of that gene's products. These consequences may affect many areas.

More detailed investigations into the gene's function can be determined by introducing much more subtle changes, which will hopefully affect only one specific area. For example, if a certain gene were responsible for the creation and operation of a certain set of nerve cells, knocking it out would completely eliminate its neurons development.

In the 1970's, Mario Capecchi was experimenting with using glass needles which were quite tiny, to inject DNA directly into the nuclei of the cells of mammals. He found this procedure to be quite good, and usually between three and five cells would receive the DNA in a functional form and subsequently pass it on to their daughter cells.

When Capecchi followed the fate of these cells, he noticed an interesting phenomenon. More than one of these new randomly inserted DNA molecules could be inserted at the same site, and they would all line up in the same direction. Capecchi and his co-workers went on to show that cells use a process called homologous recombination to stick such molecules together . This process only works on DNA molecules with the same nucleotide sequence however. These molecules line up next to one another, are cut, and then are joined at the cut ends. This joining is so precise that no nucleotide sequences are altered.

Capecchi further found that even if he injected 100 DNA molecules of the same sequence, they would still all be stitched together in the same direction. Capecchi then realized that if the could get this process to occur between a new piece of DNA and its counterpart in the cell, then one could rewrite the cell's instruction book at will.



Examples of Targeted Gene Replacement

One example of targeted gene replacement in mice deals with a gene called lpr or Fas. This gene was understood to play a role in apoptosis. Apoptosis is a process that enables the body to get rid of damaged or potentially harmful cells. If this gene and others are defective, a lupus-like autoimmune disease is produced in the mouse. In humans, lupus produces painful inflammations in different parts of the body such as the joints, skin, kidneys, lungs, heart, nervous system, and blood vessels. The disease ranges from a mild case to one that can be life-threatening, and it primarily affects woman of childbearing age.

In mice homozygous recessive for this gene, production of the Fas apoptosis protein is reduced, and many characteristics of lupus found in humans are noted. These mice also develop lymphoproliferative disease, which is production of an abnormally high number of lymphocytes.

Apoptosis is responsible in the immune system for eliminating white blood cells with a potential to attack the bodies own tissues. However, if the Fas gene is defective, these dangerous cells will survive and go on to produce autoimmune diseases like lupus. Some of these cells will also produce autoantibodies, which are antibodies directed against the body's own cells.

However, when this defective gene is replaced, the lupus-like symptoms in the mice disappear and the mice no longer show signs of the disease. Research has suggested that an apoptosis defect also occurs in humans with lupus, and there is hope that the gene-targeted cure in mice can someday be harnessed so that it will be applicable to humans.

A further example of targeted gene replacement works sort of backwards. Once again, mice were used. The gene geneticists wished to study was one responsible for Gaucher Disease, which is a fat storage disorder. This time, researchers replaced a normal gene with a defective one, in order to replicate symptoms of the disease found in humans in mice. Scientists thus hoped to test therapies for the disease on mice.

Homozygous recessive mice or humans have a defective form of an enzyme that normally breaks down fats in certain body tissues. Fatty waste products thus build up in the cells and this can cause and enlarge spleen and liver, bone deterioration, and brain abnormalities. While infusions of normal enzymes represents one form of treatment, it is quite costly. To treat an adult with the disease for one year, the cost is around $200,000. These Gaucher mice thus represent a way to test new enzyme replacement treatments.

In conclusion, targeted gene replacement provides science with a novel new way to treat various disorders and abnormalities. While this type of research may lead to powerful new ways to combat diseases, one has to wonder if science will stop here. Or will gene replacement continue, until it is possible to replace genes that can change one's appearance? How many of us would want to partake of such a service if it would make us taller or change the color of our hair? Will gene replacement provide us with a way to tailor make our looks sometime in the future? This is a question that will only be answered in time.



Sources: http://www.nih.gov/niams/news/fasgene.htm Contact: Elia T. Ben-Ari Received: 12-03-97

http://www.os.dhhs.gov/news/press/pre1995pres/920603.txt Contacts: Jules Ashen, Michaela Richardson Received: 12-03-97

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