Biotechnological Methods and Products

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5 Δεκ 2012 (πριν από 4 χρόνια και 7 μήνες)

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Applications of Biotechnology

Recombinant DNA

Products and Organisms

Applying Your Knowledge

Applying Your Knowledge

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 ___________.

1.
Probe

2.
Clone

3.
Plasmid

4.
Restriction Enzyme

Recombinant DNA Products

Pharmaceutical

Used for

Factor VIII

Blood Clotting

Human Growth
Hormone

Pituitary
Dwarfism

Insulin

Diabetes

Interferon

Cancer

Tissue Plasminogen
Activator

Heart Attack

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

Advantages

Vector

Disadvantages

Plasmid

Cosmid

(from

bacterial virus)

Virus

Can carry foreign DNA
into bacteria and yeast

Must remain small in
size to increase uptake
and avoid damage

Can carry large
DNA fragments

Technically more
difficult to construct than
recombinant plasmid

Can reach plant and
human cells

Disabled virus may

regain disease
-
causing properties

Selecting the Host Cell



Genetics is well


known


Plasmids and


viruses are well


known


High reproductive


rate


Can cause disease


Endotoxins from


cell wall


Poor protein exporter


Cannot remove introns

Advantages

Prokaryotic

Host

Disadvantages

Bacillus

E. coli


Actively exports


proteins



Plasmids and


viruses are well


known


Cannot remove introns

Selecting the Host Cell



Genetics is well


known



Non
-
pathogenic



Proteins of greater


complexity can


be made


Removes introns

Advantages

Eukaryotic

Host

Disadvantages

mammalian

yeast


Avoids toxic


bacterial proteins


Allows proper export,


protein folding


and modification


EXPENSIVE!


May not be able to
modify or export
complex mammalian
proteins

Applying Your Knowledge

1.
Plasmid

2.
Cosmid

3.
Virus

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)

4.
Yeast

5.
mammalian cell

6.
E. coli

Microinjection:
Placing DNA in each
cell with a thin
needle (animal cells,
plant protoplasts)

DNA Delivery

Methods

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

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

Coding

Region

Plasmid Sequences

-
origin of replication

-
antibiotic resistance



Ribosome

Binding Site

for mRNA

Promoter

-

Binding site for


RNA polymerase

-

Initiation of


transcription

Terminator

-
Site where

transcription
ends

Expressing the Gene: Production of
Protein


Increased

Yield

Goal

Possible Methods



To efficiently


produce as much


protein as possible



Increase plasmid


copy number


Increase


promoter


efficiency


Increase mRNA


binding to


ribosome

Collecting the Gene Product



less protein


breakdown



No cell can be


protease
-
free

Advantages

Disadvantages

Avoiding Protein

Loss using


Protease
-
deficient

Bacteria


Fusion to native


proteins




Accumulation of

aggregates


proteins not


degraded


avoids protease


activity



May be technically


difficult


Must remove native


protein


Aggregates difficult


to extract without


breakdown

Collecting the Gene Product



Used to increase
export of a protein


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

How it Works

Addition of

a Signal

Sequence

Uses


Used to Treat:



Dwarfism

Human Growth
Hormone


Functions:

-
Increases cell


uptake of amino


acids

-
Promotes use


of fat



Advantages: Decreased danger of disease

Less expensive, available in larger quantities

Pharmaceutical Products


Size

191 Amino Acids


Library?


Vector?


Host?


cDNA


plasmid


bacterial


(after removing


signal sequence)


Used to Treat:



Hemophilia

Factor VIII


Function:


-
Blood Clotting




Advantages: Easier to obtain


Free from contamination

Pharmaceutical Products


Size


-
2332 Amino Acids


-
26 introns


-
25 carbohydrate


attachment sites


on the protein


Library?


Vector?


Host?


cDNA


virus


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

Product

Action

Goats

tPA = tissue

Plasminogen
Activator

Breaks up blood
clots to treat
Heart Attacks

Pigs

Hemoglobin

Blood Substitute

Sheep

Alpha
-
1
-
antitrypsin

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.
Knockout Animal

2.
“Pharm” Animal

3.
Model of Human Disease

4.
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