Molecular Genetics

Other Pages

A variety of DNAs (and some RNAs) have been modified to carry added genes into eucaryotic cells and organisms. Among the more important vectors are those derived from retroviruses and the Agrobacterial Ti plasmids. Others are derived from viruses such as vaccinia virus and Baculovirus and from P transposable elements.

Vaccinia virus

• Vaccinia virus has been used for immunization for about 200 years.
• Its 187 kbp linear DNA has more than 12 sites where additional DNA can be inserted.
• Insertion of genes in these sites is by recombination at flanking homologies (see diagram for baculovirus).
• The major use of vaccinia virus vectors is as antigen delivery vehicles for immunization.

Baculovirus

• Baculoviruses infect insect cells. Their large (128 kbp) double-stranded genomes have a gene for a protein, polyhedrin, that is made in large amounts in infected cells.
• Polyhedrin is only needed for dissemination of the virus in nature.
• Small plasmid vectors containing regions of baculovirus DNA taken from either side of the polyhedrin gene are engineered to contain a gene to be transferred (YFG) to a baculovirus.
• Cotransfection of insect cells with the engineered plasmid and wild type baculovirus DNA results in recombination. Recombination creates baculovirus DNA with YFG replacing the polyhedrin gene.
• Baculoviruses are used primarily to produce large amounts of protein in eucaryotic cells, but can also be used for biocontrol of insect pests.

P elements

• The P element of Drosophila is a transposable DNA element, a segment of DNA encoding machinery for movement of DNA from one location to another.
• Which DNA is moved is determined by sequences at the ends of the targets. Target specificity is determined by the transposase synthesized from a gene in the element.
• Fly embryos are microinjected with intact P elements and YFG flanked by target ends.
• Resulting flies have a single copy of YFG integrated into the chromosome.
• P element transformation is used to test whether isolated genes will complement known mutations.

Implications and Further Information:

• Mobile genetic elements and viral genomes can be manipulated to deliver genes to cells.
• Homologous recombination is used to insert payloads into larger vectors.
• Recipient cells may either have extra DNA as an episome (producing transient expression) or have the DNA integrated into their chromosomes (producing transgenic cells).