|MadSci Network: Genetics|
The insertion of DNA (deoxyribonucleic acids) into living cells is the hallmark of molecular biology and genetic engineering. There are several techniques for getting DNA into cells, depending on the types of cells, whether the cells are from bacteria or higher eukaryotes (i.e. plants, animals, or fungi), and the types of DNA.
Two techniques can be used to get DNA into bacteria, infection and transformation. Infection involves linking the sample DNA to the DNA of a virus called a bacteriophage, phage for short. The normal activity of a phage is to attach itself to a bacterium, and inject its DNA into the cell. Any DNA spliced onto the phage chromosome is carried into the cell with the phage DNA. Transformation involves linking the sample DNA to a circular piece of DNA called a plasmid, and then allowing the bacteria to absorb the DNA through a process originally described by Fred Griffith in 1928. In the lab, we increase the rate of transformation by pretreating the bacteria to make them "competent".
For higher eukaryotes:
The two bacterial techniques, can also be applied to other cells. Viruses (like Adenovirus in humans or TMV in tobacco plants) can be used to carry DNA into some cells, but each there are more types of cells than engineered viruses, so most labs use a process similar to bacterial transformation to get DNA into their cells. There are specially designed plasmids for transforming yeast cells and plant cells. In animal cells, transformation has a different meaning (related to cancer), so we use the term transfection for the insertion of plasmid DNA into these cells. Specialized plasmids for transfection of animal have been engineered to the DNA, and several ways of getting the plasmids into the cells have been devised. The simplest is Calcium Phosphate Precipitation, in which the DNA is precipitated onto salt granules which are "eaten" by the cells. This has a relatively low transfection efficiency, so many labs use special fat droplets to carry the DNA into the cells. Some cells are resistant to these methods, so Electroporation was developed to use a strong electric current to force the negatively charged DNA into the cells. Some cells, like plant cells, have tough cell walls that prevent DNA of any kind from getting into the cells. For these cells, the DNA is coated on microscopic beads and shot at the cells from a miniature shotgun. Obviously, this technique can hurt the cells, so it is reserved for special cases.
The final technique is microinjection. Microinjection is usually reserved for making Transgenic Mice, mice with new genes added to their cells. To make mice transgenic, special Embryonic Stem cells are grown in culture, and then individual cells are injected with DNA through microscopic glass needles. Then the cells are mixed with normal embryonic cells and injected into the womb of a female mouse to develop into a mouse containing the new DNA. This is a very difficult and time consuming technique, which usually requires taking a course and reading a manual, or sending the DNA to another laboratory which will insert the DNA and grow the mice for you.
Molecular Biologists use almost all of these techniques to study how genes work, where genes are on the chromosomes, and how different genes interact.
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