Transformation, stable

C. R. Barton and co-workers, "Stable Transformation of Foreign DNA into Coffea Arabica Plants," in Ref. 40. 

Ketchum, R.E.B., Wherland, L. and Croteau, R.B. (2007) Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures. Plant Cell Reports, 26, 1025-1033. 

Agrobacterium-mediated stable transformation -Plant Agrobacterium-mediated stable transformation -Cell culture Transient protoplast transformation Particle bombardment-based transient expression Agrobacterium- mediated transient expression... 

Although not as popular as the baculovirus-system, stable transformations of insect cells can be used to circumvent the problems mentioned in the last paragraph. Common hosts include the fruitfly and mosquito. The expressed genes are often under the control of the Drosophila metallothionein promoter. Genes coding for resistance to antibiotics such as hygromycin and neomycin are used as selection markers. 

The stable transformation of plants has been used routinely as a method to express vaccine and therapeutic proteins. A number of disadvantages such as the length of time taken for transgenic plants to be generated and containment (i.e., preventing the escape of transgenes into the environment) have brought about a search for alternative methods by which to express proteins in plants. One such alternative is the utilization of plant virus expression systems. 

Michael Fromm, molecular biologist al the Plant Gene Expression Center, reported the stable transformation of com using a high-speed gene gun. 

Titomirov, A.V., Sukharev, S. and Kistanova, E. (1991) In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim. Biophys. Acta., 1088,131-134. 

McCabe, D., Swain, W., Martinell, B. and Christou, P. (1988) Stable transformation of soybean (glycine max) by particle acceleration. Bio/Technology, 6, 923-926. 

QM are the final stable transformation products of phenolic antioxidants 11-14,16. Formation of QM cannot be avoided in phenols containing a methyl or substituted... 

Two days after transfection, add hygromycin to a final concentration at 200 pg/mL to obtain stable transformants. When the cells grow nearly confluent, dilute the culture 3-5x by adding RPM II640/10% FCS/200 pg/mL hygromycin. Continue culture under the selection with 200 pg/mL hygromycin at least for a week and until total cell number becomes over 2 x 107. 

In our experience, about 106 stable transformants were routinely obtained from 107 Raji cells. Therefore, in order to obtain a population that represents the full cDNA library, perform one electroporation with 107 Raji cells/2 x 105 independent E. coli colony. 

Campoy, S., Perez, F., Martin, J. F., Gutierrez, S., and Liras, P. 2003. Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer. Curr. Genet. 43(6), 447-452. 

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