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
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
Bacteria
Production of Penicillin
Penicillin first discovered, produced in small quantities.
Attempts to increase penicillin production during WWII.
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.
Purification of recombinant proteins
Transgenic technology as an alternative to
drug-producing cells
Transgenic Technology
Introduction of genes (including human genes) into the germ-line 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)
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.
Steps in Transgenic Technology
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.
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.
How may mammary tissue be used to produce recombinant protein?
Human Protein C
Blood protein.
Functions to control blood clotting.
Some individuals have inborn deficiency
require exogenous Protein C.
DNA fragment containingnew hybrid gene
Human sequencefor protein of
interest (i.e.drug)
Mouse promotersequence for a milk protein
Collectionof pig embryos
MalePronucleus
FemalePronucleus
“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
Egg
Discardnucleus Enucleated ova
(no nucleus)Egg containing new nucleus
A CLONE
Nucleus removed and injected into enucleated egg(nuclear transfer)
Parental cell
How’s about we clone some Mice??
Bleecker-o-Matic CloneAll 9000
Animals Cloned (as of early 2003)
SheepCowPigGoatMouseCatRatWild 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 foreigngene, that would be beneficial.