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Why Recombine DNA?
1. To produce protein products
2. To alter genetic inheritence (new traits)
3. For diagnostic tests – allows researchers to study causes
of genetic or infectious disease
Systems to express recombinant proteins
Bacteria
Yeast
Mammalian cells
Also….
Plants
Insect cells
Transgenic animals
Bacteria
E. coli most common strain
Advantages
Rapid growth on low-cost media
Easy to scale-up from lab to production
Disadvantages
Proteins produced in E. coli are not glycosylated.
Expressed protein may aggregate or fold improperly
Production of Penicillin
Penicillin first discovered, produced in small quantities.
Attempts to increase penicillin production during WWII.
Purification of recombinant proteins
1) Purify the recombinant drug from mix of proteins in
the producer cell.
2) Remove contaminants that may be present in
bacteria, mammalian cells or serum.
Transgenic technology as an alternative to
drug-producing cells
Transgenic Technology
Introduction of genes (including human genes) into the germline cells of plants and animals.
Provides stable introduction of foreign genes at the
embryonic level.
Transformed organisms will pass along the new genes to
their offspring.
Why Transgenic Technology?
Study human diseases using a transgenic animal model
Use transgenic animals or plants to produce a desired
product (e.g., drugs)
Steps in Transgenic Technology
Insert gene of interest into the nucleus of a fertilized egg.
Implant into female mouse.
Isolate DNA from each of the offspring to determine which
offspring carries the transgene.
Continuous matings to produce a stable transgenic line.
Green Fluorescent Protein (GFP)
Normal mice
Transgenic mice with GFP
Tobacco
Plant
Transgenic animals as drug factories
Desirable to have the protein drug secreted in an easily
retrievable manner.
A recombinant drug, secreted into the milk of the transgenic
animal could be produced in large quantities and easily
retrieved from the animal.
How may mammary tissue be used to
produce recombinant protein?
Attach the promoter sequence of a major milk protein
upstream of the drug gene.
Although this foreign gene will be present in all of the
cells of the transgenic animal it will only be expressed
in the mammary tissue.
Human Protein C
Blood protein.
Functions to control blood clotting.
Some individuals have inborn deficiency
require exogenous Protein C.
DNA fragment containing
new hybrid gene
Human sequence
for protein of
interest (i.e.drug)
Mouse promoter
sequence for
a milk protein
Collection
of pig embryos
Male
Pronucleus
Female
Pronucleus
“Genie” The first genetically engineered
animal to produce a human protein drug
(human protein C) in her milk.
Genie
Produced sufficient quantities of Human Protein C.
1 g of Human Protein C per 1 liter of milk.
200-times more than present in human blood.
Some examples of therapeutic protein production using
transgenic animals
Growth hormone (gigantism / dwarfism
-- goat
Human fertility hormones
– cow
Fibrinogen – for burn patients
-- sheep
Cloning = Asexual Reproduction
Whole nucleus of any cell type is used.
Children are genetically identical to parent.
All offspring (children) carry same genetic material.
Process of Cloning
Parental cell
Egg
Discard
nucleus
Enucleated ova
(no nucleus)
Nucleus
removed and
injected into
enucleated egg
(nuclear transfer)
Egg containing new nucleus
A CLONE
How’s about we clone some Mice??
Bleecker-o-Matic CloneAll 9000
Animals Cloned (as of early 2003)
Sheep
Cow
Pig
Goat
Mouse
Cat
Rat
Wild sheep
Dolly - The First Cloned Sheep
Dolly was an identical genetic copy of her mother.
A technological breakthrough.
From a pharmaceutical perspective, not very useful.
However, if one could make a clone that expressed a foreign
gene, that would be beneficial.