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.