Copyright avigen.com Vectors After the scientist has decided what gene they want to change and in which cells, they need to construct the DNA sequence that will encode for the gene they want to insert. This is done with a gene sequencer. One the piece of DNA is constructed it needs to be placed in cells, either in vitro (in the lab) or in vivo (literally "in body"). Vectors are the protocols and materials used to deliver the DNA to the cells that the scientists want to change. The main types of vectors in use today are viral and non-viral. Many viral methods also use the non-viral methods. Viral Vectors All viral vectors are viruses changed to make them perfectly harmless. The DNA to be delivered is then inserted into the viral DNA. The viruses are then put in the body. Viruses are used because they naturally go through the body and inject their viral DNA into the host's cells. The viral DNA is then part of the host cell's DNA and the cell replicates taking the new DNA with it. That at least, is the plan. All viral vectors have the chance of somehow recreating the wildtype (natural) virus which could then act in unpredictable ways. Retroviral Vectors Retroviral vectors use retroviruses (one of the most famous retroviruses is HIV) to infect the host cells with the new gene. The positive aspects of retroviruses are the fact that they hold large amounts of genetic material, the high rate of insertion of the new gene into the host's cells, and the fact that the vector does not add its own wildtype DNA to the host's cells. The negative aspects of retroviral vectors are, the low success rate, the fact that retroviruses only infect rapidly multiplying cells, and that the location of insertion of the new gene is random (which affects how the gene manifests itself as a phenotype). Adenoviral Vectors Adeno viruses are what cause the common cold. They locally affect the membranes and tissues of the eyes and respiratory system, and they affect all the cells of those areas, whether quickly or slow replicating cells. These vectors can only affect a cell one time. Unfortunately the inserted DNA does not become part of the host cell's DNA. The host also kills off these viruses, usually long before they reach their target. Adeno-Associated Viral Vectors Adeno-associated viral vectors can affect the central nervous system in a semipermanent fashion. The main problem is these viruses are ones such as the Herpes Simplex Virus that can cause diseases in humans. These viruses have trouble reaching their target cells and they are less safe, because they could revert to the unsafe wildtype viruses and infect the host with herpes. Vaccinia Vectors Vaccinia vectors are based on the vaccinia virus that was used to immunize against smallpox. This option is more dangerous because one in 50,000 people has a bad reaction to this virus. It also could not be used on people who had receive the smallpox vaccination or who have weak immune systems. Non-viral Vectors Non-viral vectors are various methods of getting DNA into cells without having to use viruses. Calcium Phosphate Transfection Calcium phosphate transfection has a low rate of success, but is very cheap. DNA is put in a solution of calcium chloride. The calcium and the phosphate in DNA react and precipitate, bringing the DNA with them. That precipitate is then put with live cells. The cells take in the calcium phosphate and the DNA with it. Microinjection Microinjection is one of the methods used when creating transgenic animals. A cell is placed under a microscope and manually injected with DNA. At this time the DNA is usually injected into a fertilized somatic cell, and this has successfully created animals with DNA that humans put into the animal's genome. Electroporation Electroporation uses the methods of electrophoresis to put DNA in cells in methods when in vitro situation is acceptable. Basically, a suspension of cells and DNA is placed in a gel and a voltage is applied. That voltage runs the DNA into the cytoplasm of the cells. Sometimes this method work well, but it is very dependant of voltage and DNA size. It also has no in vivo applications at this time. Liposomes Liposomes are also being used as vectors. In regular cells liposomes are a means of transport for proteins and DNA from cell to cell as well as within the cell. Liposomes are basically packages created by pockets of membrane similar to that of the cellular membrane. They are therefore uniquely suited to carry DNA to cells and interact with either positively or negatively charged parts of the cell membrane. Positively charged areas of lipofectin, a major component of liposomes, are attracted to the negative DNA and they attach to it. If enough lipofectin molecules attach to or trap the DNA and form a liposome which can then be delivered to the body. Naked Plasmid DNA Injection This method is by far the simplest. DNA in a solution meant to keep the DNA stable is injected directly into muscle tissues. In various tests on animals this method has resulted in phenotype and in some cases short-lived genotype changes in the thymus, skin, and striated muscles. Unfortunately, all the new injected DNA did not replicate the same as regular DNA, and in at the most 19 months, the effects and traces of new DNA disappeared in all published trials. Ballistic DNA Injection Ballistic DNA injection has actually accomplished short term changes in phenotypes exhibited in both animals and plants. This method is used both in vitro and in vivo. In humans it has been used more for affecting the DNA of skin and muscles then any deeply set organs, although the liver has been used as a test organ. DNA is attached to tiny amounts of gold or tungsten, and those small particles are shot at the target area. Unfortunately this method can damage the cells of the targeted organ. Scientists are attempting to use this method to immunize against viruses like HIV. Drug Disposition Scientists also think that DNA could be put into the body in a drug like form. The problem with this method is that the DNA could be destroyed along the path it must travel to the targeted cells. The bigger problem involves the way that drugs travel through the body. First, the drug must be absorbed by a membrane in the nose, stomach, intestine, or somewhere else. Once the drug enters the bloodstream it could be absorbed by either the kidneys or the liver (all of which clear waste from the bloodstream) or it could attach itself to other molecules in the bloodstream, making the drug too large to enter the capillaries and/or get into cells. These issues are making the delivery of DNA through drugs a much trickier process. Targeting certain cells In order for gene therapy to work well and be useful, doctors and scientists need to be able to send the new DNA to the cells that need to be affected. Right now many possible protocols are under scrutiny and experimentation. One such protocol is transductional targeting, which works by finding the cells that multiply at a rapid rate. That method uses retroviral vectors, like HIV, that naturally target the white blood cells, which multiply quickly. Other methods target cells based on the protein s contained in their membranes and by attaching molecules to the DNA such that the cell recognizes the molecule as good and brings the molecule and DNA into the cell. All the information on vectors was found at the website http://www.mc.vanderbilt.edu/gcrc/gene.htm. The material was written by Jeff Fritz of Vanderbilt and was dated January 22, 1996 and January 29, 1996. The material used was found and used on March 10, 2000.