Transcript Biotechnological Methods and Products

Applications of Biotechnology
Recombinant DNA
Products and Organisms
Applying Your Knowledge
1.
2.
3.
4.
Probe
Clone
Plasmid
Restriction Enzyme
A. An enzyme that cleaves DNA at specific
sequences is a __________ .
B. A sequence of DNA that is complementary to
the gene of interest is a _________.
C. A small, independently replicating DNA
molecule is a ___________.
Recombinant DNA Products
Pharmaceutical
Factor VIII
Human Growth
Hormone
Insulin
Interferon
Used for
Blood Clotting
Pituitary
Dwarfism
Diabetes
Cancer
Tissue Plasminogen Heart Attack
Activator
Vaccine
Hepatitis B
Optimizing the Yield
of a Gene Product
•
•
•
•
Selecting the Vector
Selecting the Host Cell
Selecting the DNA Delivery Method
Constructing the Recombinant DNA
Molecule
• Maximizing Gene Expression
• Collecting the Gene Product
Vectors
Plasmids
Shuttle vectors
Viruses
Artificial Chromosomes
Expression Vectors
Selecting the Vector
Vector
Plasmid
Cosmid
(from
bacterial virus)
Virus
Advantages
Can carry foreign DNA
into bacteria and yeast
Can carry large
DNA fragments
Can reach plant and
human cells
Disadvantages
Must remain small in
size to increase uptake
and avoid damage
Technically more
difficult to construct than
recombinant plasmid
Disabled virus may
regain diseasecausing properties
Selecting the Host Cell
Prokaryotic
Host
E. coli
Bacillus
Advantages
•Genetics is well
known
•Plasmids and
viruses are well
known
•High reproductive
rate
•Actively exports
proteins
• Plasmids and
viruses are well
known
Disadvantages
•Can cause disease
•Endotoxins from
cell wall
•Poor protein exporter
•Cannot remove introns
•Cannot remove introns
Selecting the Host Cell
Eukaryotic
Host
yeast
mammalian
Advantages
Disadvantages
•Genetics is well
known
• Non-pathogenic
• Proteins of greater
complexity can
be made
•Removes introns
•May not be able to
modify or export
complex mammalian
proteins
•Avoids toxic
bacterial proteins
•Allows proper export,
protein folding
and modification
•EXPENSIVE!
Applying Your Knowledge
1. Plasmid
2. Cosmid
3. Virus
4. Yeast
5. mammalian cell
6. E. coli
A. Best for large sequences of DNA (1-3)
B. Best for proper modification of complex
proteins (4-6)
C. Best for entry into specific cell types (1-3)
D. Inexpensive organism that can remove
introns (4-6)
Virus: Binds to a
receptor on the cell
surface and injects
DNA into the cell
(bacteria, plants,
animals)
Liposome:
Sphere of lipids
that is compatible
with plasma
membrane
(animal cells,
plant protoplasts
DNA Delivery
Methods
Microinjection:
Placing DNA in each
cell with a thin
needle (animal cells,
plant protoplasts)
Additional DNA Delivery Methods
Method
How it Works
Used For/In
Chemicals that open
holes in membranes
Temporary holes admit DNA
Bacterial cells
Animal cells
Plant protoplasts
Electroporation
Electric shock causes
temporary holes to admit DNA
Animal cells
Plant protoplasts
Particle
Bombardment
DNA-coated metal pellets are
propelled into cells with “gene
gun”
Plant cells
Constructing the Recombinant DNA Molecule
Ribosome
Binding Site
for mRNA
Coding
Region
Promoter
- Binding site for
RNA polymerase
- Initiation of
transcription
Plasmid Sequences
-origin of replication
-antibiotic resistance
Terminator
-Site where
transcription
ends
Expressing the Gene: Production of
Protein
Goal
Increased
Yield
• To efficiently
produce as much
protein as possible
Possible Methods
• Increase plasmid
copy number
•Increase
promoter
efficiency
•Increase mRNA
binding to
ribosome
Collecting the Gene Product
Avoiding Protein
Loss using
Advantages
Disadvantages
Protease-deficient
Bacteria
• less protein
breakdown
• No cell can be
protease-free
Fusion to native
proteins
•proteins not
degraded
• May be technically
difficult
•Must remove native
protein
Accumulation of
aggregates
•avoids protease
activity
•Aggregates difficult
to extract without
breakdown
Collecting the Gene Product
How it Works
Addition of
a Signal
Sequence
•A signal sequence
directs a ribosome to
attach to rer to
complete synthesis of
the protein
•Protein product is
delivered into rer for
transport within the
cell to the outside
Uses
• Used to increase
export of a protein
Pharmaceutical Products
Human Growth
Hormone
Functions:
-Increases cell
uptake of amino
acids
-Promotes use
of fat
Used to Treat:
Dwarfism
Advantages: Decreased danger of disease
Less expensive, available in larger quantities
Size
191 Amino Acids
Library?
cDNA
Vector?
plasmid
Host?
bacterial
(after removing
signal sequence)
Pharmaceutical Products
Factor VIII
Advantages: Easier to obtain
Free from contamination
Function:
Size
-Blood Clotting
-2332 Amino Acids
-26 introns
Used to Treat:
Hemophilia
-25 carbohydrate
attachment sites
on the protein
Library?
cDNA
Vector?
virus
Host?
mammalian
(hamster)
Transgenic Animals
• Transgenic = containing one or
more genes from another species in
every cell of the organism
• Types
1. “Pharm” Animals
2. Models of Human Diseases
3. Gene Targeted Animals
“Pharm” Animals
• Animals that produce pharmaceuticals
and release them in milk or semen
Goats
Pigs
Product
Action
tPA = tissue
Plasminogen
Activator
Hemoglobin
Breaks up blood
clots to treat
Heart Attacks
Sheep Alpha-1antitrypsin
Blood Substitute
Hereditary
emphysema
Models of Human Diseases
• Transgenic Mice act as Models of
Human Diseases for Research
Human Gene Mouse Model For
BRCA1
Breast Cancer
HD
Huntington’s Disease
HGH
Human Patients
Receiving Human
Growth Hormone
Procedure for Gene Targeting
Gene Targeting
• Introducing a different form of a gene to
replace a host gene
“knockout”
Inactivation of a gene by gene
targeting
---mouse with ADA gene knocked out as
a model for SCID
“knock in”
Introducing a gene with altered
function by gene targeting
---mice with
human adult hemoglobin
human fetal hemoglobin
human sickle cell hemoglobin
Applying Your Knowledge
1.
2.
3.
4.
Knockout Animal
“Pharm” Animal
Model of Human Disease
Knock in Animal
A. Animal that secretes medicine in its milk
B. Mouse with a human BRCA 1 gene added
(no mouse genes altered)
C. Mouse with its ADA gene inactivated