Cloning Vectors for Eukaryotes
Vectors for yeast
The 2 µm plasmid is a naturally occuring 6 kb episomal plasmid found in yeast.
Selection is based on nutrition instead of drugs. Yeast cells that are auxotrophic (unable to synthesize an essential component, like an amino acid) can only grow when nutritionally supplemented, or when the missing nutritional function is provided for by genes contained on a plasmid or extra chromosome.
Many eukaryotic vectors contain both eukaryotic and prokaryotic signals and can be replicated in both types of cells. These are called shuttle vectors.
Often, much of the cloning is done in prokaryotic systems and then the recombinant vectors are grown in eukaryotic cells.
Why not just express all genes in prokaryotes? Why transfer to eukaryotes for expression?
YEp Yeast episomal plasmid
YIp - Yeast integrative plasmid
YRps - Yeast replicative plasmid
YACs - Yeast artificial chromosome (600-1400 kb capacity)
Cloning vectors for higher plants
Agrobacterium tumefaciens
The system was derived from the bacteria that causes crown gall disease in plants.
During infection, the Ti plasmid is integrated into the plant chromosomal DNA.
The functions of the Ti plasmid have been divided between two plasmid-like vectors which are co transformed and operate as a binary vector system.
Transformed plants can be recovered.
A binary vector
Direct gene transfer randomly integrates supercoiled DNA into cells (no homology required).
Cloning vectors for animals
Cloning into insect cells
Drosophilia cells and the P vector
Baculovirus
Cloning in mammals
Purposes for cloning into animals
Gene knockout
Production of recombinant proteins
Gene therapy
Vectors are often derived from viral components
