Cloning in eukaryotes

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CLONING IN EUKARYOTES
SACCHAROMYCES CEREVISIAE
PICHIA PASTORIS
By
Priyengha R S

GENE CLONING
 Gene cloning is the process of incorporating
foreign genes into hybrid DNA replicons.
 Cloned genes can be...

DNA CLONING
 Restriction enzymes (endonucleases):
in nature, these enzymes
protect bacteria from intruding DNA; they
cut ...

STEPS FOR EUKARYOTIC GENE
CLONING
 Isolation of cloning vector
(bacterial plasmid) & gene-
source DNA (gene of interest)
...

Restriction
endonucleases

WHY CLONE IN EUKARYOTES?
 Eukaryotic genes may not be expressed properly in
bacterial host
 different mechanisms for gen...

Chapter11GeneticEngineering:Gene
Cloning

TRANSFECTION OF
EUKARYOTIC CELLS
Transfection originally referred to
transformation of viral nucleic acid into cells
(bot...

SHUTTLE VECTORS AND EXPRESSION
VECTORS
 Shuttle Vectors
 Vectors that can replicate and are stably
maintained in two (or...

Genetic map of a shuttle vector
used in yeast

Expression Vectors
 are designed to allow the experimenter to
control the expression of cloned
 contain regulatory sequ...

Genetic map of the expression
vector pSE420.

EUKARYOTIC VECTORS
It is often desirable to clone and express
genes directly in eukaryotes, and vectors
are available for...

BACTERIOPHAGE LAMBDA AS A
CLONING VECTOR
Lambda can also be used as a cloning
vector for in vitro recombination
useful c...

MODIFIED LAMBDA PHAGES
Wild-type lambda is not suitable as a
cloning vector because its genome has too
many restriction e...

EUKARYOTIC ARTIFICIAL CHROMOSOMES
 Yeast Artificial Chromosomes (YACs)
 first artificial chromosomes
 replicate in yeas...

Human artificial chromosomes (HACs)
Have also been developed and are
similar to YACs in overall structure.
Circular*
H...

• The choice of an expression system depends
primarily on the quality of the recombinant protein.
• However, the yield of ...

• The major features of a eukaryotic expression vector
are a promoter, a multiple cloning site, DNA segment
for terminatio...

• Saccharomyces cerevisiae
• Pichia pastoris
• Baculovirus-insect cell lines
• Mammalian systems
EUKARYOTIC EXPRESSION SYS...

• It is the most common eukaryotic system and
there is a great deal of study about this organism.
• It is a single-celled ...

• There are three main classes of S. cerevisiae
expression vectors.
• Yeast episomal plasmids (YEps).
• Yeast integrating ...

• The YEps vectors are based on the high-copy-
number 2µm plasmids.
• The vectors replicate independently via a single
ori...

• Generally, tightly regulatable, inducible promoters are
preferred for producing large amounts of recombinant
protein at ...

• Most heterologous genes are provided with a
DNA coding sequence for signal peptide that
facilitates the secretion of pro...

• Plasmid-based yeast expression systems are often
unstable under large-scale growth conditions even
in the presence of se...

Integration of DNA with a Yip vector

YEAST ARTIFICIAL CHROMOSOMES
 YAC is an artificially constructed chromosome that
contains a
 Centromere
 Telomeres
 Au...

YACs are plasmid shuttle vectors capable of
replicating and being selected in common
bacterial hosts such as Escherichia c...

Purpose of using YAC Vectors:
Cloning vehicles that propogate in eukaryotic cell hosts as
eukaryotic Chromosomes
Clone ver...

CONSTRUCTION OF YEAST ARTIFICIAL
CHROMOSOMES
Plasmid DNA purification
Treatment with restriction
enzymes
Ligation and yeas...

• A YAC is designed to clone a large segment of DNA
(100 kb), which is then maintained as a separate
chromosome in the hos...

YAC cloning system

• Human Cu/Zn SOD cDNA was cloned between the
promoter and termination-polyadenylation sequence of
the yeast GAPD gene and...

• Proteins may also be produced for secretion.
• In this system, any glycosylated protein is secreted (O
or N-linked).
• T...

• For example, a properly processed and active form of
the protein hirudin; a powerful anticoagulant protein
cloned from a...

Secretion of Heterologous Proteins

• Though S. cerevisae is successfully used to produce
recombinant proteins for human, it has major
drawbacks.
• The level ...

• P. pastoris is a methylotrophic yeast that is able to
utilize methanol as a source of carbon and energy.
• Glycosylation...

Pichia pastoris Expression Systems

Pichia pastoris Expression Systems

Pichia pastoris vs Saccharomyces cerevisiae
Advantages P. pastoris and S. cerevisiae
Short doubling time
Readily manipulat...

PRACTICAL DNA TECHNOLOGY USES
 Diagnosis of disease
 Human gene therapy
 Pharmaceutical products
(vaccines)
 Forensics...

GENES THERAPY

BIOTECHNOLOGY PRACTICAL USE

Thank You..

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