17-01-2013, 04:51 PM
YEAST & MAMMALIAN VECTORS
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INTRODUCTION
A cloning vector is a DNA molecule in which foreign DNA can be inserted or integrated and which is further capable of replicating within host cell to produce multiple clones of recombinant DNA.
Yeast Vectors
The plasmid is a naturally occurring 6 kb episomal plasmid found in yeast.
Selection is based on nutrition instead of drugs.
Yeast
• Cloning in E. coli, then transfect DNA into yeast and screen for stable integration into yeast chromosome
• Time required for selection of high expressing clones
• Once established, rapid growth in shake flasks or fermentation
Yeast – Benefits and Issues
Provides eukaryotic secretion and Glycosylation
Glycosylation is less complex than mammalian, high mannose
Saccharomyces Cerevisiae
Used for both intracellular and secreted proteins
Mammalian Vectors
More time & labor intensive than E. coli, but…
Provides most authentic secretion,
Glycosylation, phosphorylations, and other
post-translational modification
General Steps of Cloning with Any Vector
prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends
ligate the foreign DNA into the vector with the enzyme DNA ligase
introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation
select cells containing foreign DNA by screening for selectable markers (usually drug resistance)