Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

Introduction to Recombinant Genetics- Biology 350

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CLONING VECTORS FOR EUKARYOTES Vectors For Yeast The 2 m 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.

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

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.

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

Why not just express all genes in prokaryotes? Why transfer to eukaryotes for expression? YEp Yeast episomal plasmid

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

YIp - Yeast integrative plasmid YRps - Yeast replicative plasmid

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

YACs - Yeast artificial chromosome (600-1400 kb capacity)

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

CLONING VECTORS FOR HIGHER PLANTS Agrobacterium Tumefaciens The system was derived from the bacteria that causes crown gall disease in plants.

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

During infection, the Ti plasmid is integrated into the plant chromosomal DNA.

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

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.

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

Transformed plants can be recovered.

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

A binary vector

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

Direct gene transfer randomly integrates supercoiled DNA into cells (no homology required).

CLONING VECTORS FOR ANIMALS

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

Cloning Into Insect Cells Drosophilia cells and the P vector

Baculovirus

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

Cloning In Mammals Purposes for cloning into animals Gene knockout Production of recombinant proteins Gene therapy Vectors are often derived from viral components

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Cloning Vectors for Eukaryotes

http://bioweb.wku.edu/courses/biol350/cloningvecteuk9/review.html

2005 by CA Rinehart

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This material is intended for use only by WKU students registered for Biology 350. Other uses prohibited.

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