Recombinant DNA has opened new avenues for the study of gene function. The function of a cloned gene can be investigated by introducing the cloned DNA into eukaryotic cells. In simple eukaryotes such as yeasts Saccharomyces cerevisiae, molecular clones of various mutants have been prepared for genetic analysis.
Like bacteria, yeasts offer advantages in having a small genome of about 1.2 x 107 bp and a rapid rate of reproduction. To clone a mutant gene in yeast, first a genomic library of normal yeast DNA is prepared in plasmid vectors that can replicate in yeasts and in bacteria. A mixture of such plasmids is used to transform a temperature-sensitive mutant of yeast.
Such mutants encode proteins that are functional at one temperature, called the permissive temperature, but non-functional at another temperature, called the non-permissive temperature. Transformed yeast cells are easy to select because they grow at the non-permissive temperature, as they have acquired a normal copy of the gene from plasmid DNA. A number of yeast proteins have been characterised in this manner.
Genetic manipulation techniques that are possible in simple eukaryotic cells as those of yeasts are not applicable to cells of complex eukaryotes. Methods are however available for introduction of cloned DNA into cells of plants and animals, referred to as gene transfer. One common approach is to incorporate eukaryotic DNA into the genome of a non-replicating virus, followed by infection of a host cell.
Several viruses can infect a cell and integrate their genome into the DNA of host cell. If the viral genome contains an insert of foreign DNA, that would also be integrated into host cell genome. This technique of gene transfer is called transduction.
Methodologies are now available to introduce naked DNA into animal cells in culture as a co-precipitate with calcium phosphate. The process is called transfection (derived from transformation + infection). The cells in suspension exposed to a fine calcium precipitate of the DNA take up DNA and transport it to the nucleus, where it can be transcribed for several days, referred to as transient expression.
A very small fraction of cells, that is less than 0.1%, are competent to be transfected, in which the foreign DNA becomes stably integrated into the cell’s genome. Transfected cells generally pick up several fragments of DNA.
The stably transformed cells that have integrated foreign DNA can be selected by including a gene, such as a gene for drug resistance that allows transfected cells to grow, and inhibits growth of non-transfected cells.
Thus, any cloned gene can be introduced into animal cells by being transferred together with a drug resistance marker that allows stable transformants to be isolated. Transfected cells can be used to address a wide variety of questions concerning cell growth and differentiation, mechanisms that regulate gene expression and protein function.
Depending on the purpose of the experiment, various other methods are available to introduce DNA into mammalian cells. Transfection of both plant and animal cells can be carried out by electroporation. The cells are incubated with DNA in vials that contain electrodes that deliver a brief electric shock.
The electric pulse opens pores in the plasma membrane, called electroporation, that makes the plasma membrane transiently permeable to DNA molecules, some of which find their way into the nucleus and become integrated into the chromosomes, producing stably transformed cells.
Factors that influence the efficiency of electroporation include temperature, some electric field parameters such as voltage, resistance and capacitance, the topological form of DNA and some host cell factors (growth conditions and genetic background). DNA can also be incorporated into lipid vesicles called liposomes that fuse with the plasma membrane, and deliver DNA inside the cell. Direct microinjection of DNA can also be done into the cell nucleus.
A direct way to introduce foreign genes into a cell is to microinject DNA directly into the nucleus. The eggs of amphibians such as Xenopus are particularly suitable, and have been used in classical studies on giant chromosomes called lampbrush chromosomes in which transcriptional activity can be visualised.
When foreign DNA is injected into the nucleus it is readily transcribed. The RNA transcripts from the foreign DNA templates are transported to the cytoplasm, and translated into proteins that can be detected by immunological methods using specific antibodies.