Putting DNA Back into Cells
After the DNA sequence to be cloned has been joined to the appropriate vector, the hybrid must be transformed into cells for biological amplification. The phenomenon of transformation of pneumococci by DNA has been known since 1944. Once the desirable genetic properties ofE. coli were realized, it too was tried in transformation experiments. Thesewere unsuccessful for many years. Unexpectedly, a method for trans-forming E. coli was discovered. This occurred at a most opportune time, for the developments in the enzymology of DNA cutting and joining were almost ready to be used in a system of putting the foreign DNA into cells. Reintroducing a DNA molecule containing a replicon into a cell permits a biological amplification of that cell to greater than 1012. In one day, a single molecule can be amplified to quantities sufficient for physical experiments.
The key to the initial transformation protocols of E. coli was treat-ment of the cells with calcium or rubidium ions to make them compe-tent for the uptake of plasmid or phage DNA. The term for transformation with phage DNA that then yields an infected cell is transfection, and this term is used for infection of higher cells with nonvirus DNA as well. Yeast may be transfected after a treatment that includes incubation in the presence of lithium ions. Some types of mammalian cells, mouse L cells for example, can be transfected merely by sprinkling them with a mixture of the DNA and calcium-phosphate crystals. Here the mechanism of transfection appears to be uptake of the DNA-calcium-phosphate complex.
Direct manual injection of small volumes of DNA into cells has been highly useful for the study of cloned DNA fragments because it elimi-nates the need for a eukaryotic replicon or a selectable gene. Microin-jection into the oocytes of the frog Xenopus laevis has yielded much information, and microinjection into cultured mammalian cells is also possible. DNA injected into Xenopus cells is transcribed for many hours and translated into easily detectable amounts of protein. As a result of these properties, a DNA segment can be cloned onto a plasmid such as pBR322, manipulated in vitro, and injected into the cells for examination of its new biological properties. Microinjection is also possible into a fertilized mouse embryo. The embryo can then be reimplanted to develop into a mouse. Since the fragments of injected DNA will recom-bine into the chromosome, the mice are transfected by the DNA. In order that all cells in a transfected mouse possess the same genetic constitution, an injected fragment must recombine into a germ line cell. Such a mouse is unlikely to be genetically homogeneous because similar fragments probably have not integrated into somatic cells. The offspring of such a mouse will be genetically homogeneous, however, and these can be profitably studied.
Another general method for incorporating DNA into cells is electro-poration. Clls are subjected to a brief but intense electric field. This creates small holes in their membranes and for a short time DNA molecules present in the solution can be taken up.