Chp 7 Cloning Vectors for Eukaryotes Huseyin Tombuloglu PhD. GBE310, Spring 2015

The aim may not be to study a gene, To use cloning to obtain large amounts of an important pharmaceutical protein (e.g., a hormone such as insulin), or to change the properties of the organism (e.g., to introduce herbicide resistance into a crop plant). We must therefore consider cloning vectors for organisms other than E. coli. E. coli vs Eukaryotic organism

Saccharomyces cerevisiae budding yeast

As a single-cell organism, S. cerevisiae is small with a short generation time (doubling time 1.25–2 hours[19] at 30 °C or 86 °F) and can be easily cultured. S. cerevisiae divides with meiosis, allowing it to be a candidate for sexual genetics research. S. cerevisiae can be transformed allowing for either the addition of new genes or deletion through homologous recombination. Furthermore, the ability to grow S. cerevisiae as a haploid simplifies the creation of gene knockouts strains. As a eukaryote, S. cerevisiae shares the complex internal cell structure of plants and animals without the high percentage of non- coding DNA that can confound research in higher eukaryotes.

Schizosaccharomyces pombe fission yeast

Fission yeast has become a notable model system to study basic principles of a cell that can be used to understand more complex organisms like mammals and in particular humans. This single cell eukaryote is nonpathogenic and easily grown and manipulated in the lab. Fission yeast contains one of the smallest numbers of genes of a known genome sequence for a eukaryote, and has only three chromosomes in its genome. Many of the genes responsible for cell division and cellular organization in fission yeast cell are also found in the human’s genome

Fission yeast is also a practical model system to observe cell division because fission yeast’s are cylindrically shaped single celled eukaryotes that divide and reproduce by medial fission. This can easily be seen using microscopy. Short generation time, 2 to 4 hours, which also makes it an easy model system to observe and grow in the laboratory Fission yeast’s simplicity in genomic structure yet similarities with mammalian genome, ease of ability to manipulate, and ability to be used for drug analysis is why fission yeast is making many contributions to biomedicine and cellular biology research, and a model system for genetic analysis

The first patient to be treated with gene therapy was a four year old girl treated at the NIH Clinical Center in She had a congenital disease called adenosine deaminase (ADA) deficiency which severely affects immunity and the ability to fight infections. For the therapy, her white blood cells were taken from her and inserted with the correct genes for making ADA and then reinjected into her. This process was performed by Dr. W. French Anderson from the National Heart, Lung and Blood Institute.

Some antibiotics commonly used as selective agents AntibioticDescription Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by  - lactamase, which cleaves the  -lactam ring of amp Hygromycin B (HygB)Blocks translocation from amino acyl site to peptidyl site Kanamycin (Kan)Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase Neomycin (Neo)Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase Streptomycin (Str)Blocks protein initiation complex formation and causes misreading during translation Tetracycline (Tet)Binds to 30S ribosomal subunit and inhibits protein synthesis; tet r gene encodes a protein which prevents transport of tet into the cell

Vector systemHost cellInsert capacity (kb) PlasmidE. coli Bacteriophage E. coli10-20 CosmidE. coli35-45 Bacteriophage P1E. coli BAC (bacterial artificial chromosome) E. coli P1 bacteriophage- derived AC E. coli YACYeast100-2,000 Human ACCultured human cells>2,000 Cloning vectors and their insert capacities