Recombining DNA for Protein Production & Altering Genetic Inheritance: An Overview

2. Why Recombine DNA? • To produce protein products • To alter genetic inheritence (new traits) • For diagnostic tests – allows researchers to study causes of genetic or infectious disease

3. Systems to express recombinant proteins Bacteria Yeast Mammalian cells Also…. Plants Insect cells Transgenic animals

4. 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

5. Production of Penicillin Penicillin first discovered, produced in small quantities. Attempts to increase penicillin production during WWII.

6. 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.

7. Transgenic technology as an alternative to drug-producing cells

8. 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.

9. Why Transgenic Technology? Study human diseases using a transgenic animal model Use transgenic animals or plants to produce a desired product (e.g., drugs)

11. 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.

12. Green Fluorescent Protein (GFP)

13. Normal mice Transgenic mice with GFP

14. Tobacco Plant

15. 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.

16. 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.

17. Human Protein C Blood protein. Functions to control blood clotting. Some individuals have inborn deficiency require exogenous Protein C.

19. DNA fragment containing new hybrid gene Human sequence for protein of interest (i.e.drug) Male Pronucleus Mouse promoter sequence for a milk protein Female Pronucleus Collection of pig embryos

20. “Genie” The first genetically engineered animal to produce a human protein drug (human protein C) in her milk.

21. 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.

22. Some examples of therapeutic protein production using transgenic animals Growth hormone (gigantism / dwarfism -- goat Human fertility hormones – cow Fibrinogen – for burn patients -- sheep

23. Cloning = Asexual Reproduction Whole nucleus of any cell type is used. Children are genetically identical to parent. All offspring (children) carry same genetic material.

24. Process of Cloning Parental cell Egg Nucleus removed and injected into enucleated egg (nuclear transfer) Discard nucleus Enucleated ova (no nucleus) Egg containing new nucleus A CLONE

25. How’s about we clone some Mice?? Bleecker-o-Matic CloneAll 9000

26. Animals Cloned (as of early 2003) Sheep Cow Pig Goat Mouse Cat Rat Wild sheep

27. 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.