1. Recombinant DNA and its Applications
Biotechnology
Recombinant DNA and its Applications
BIOTECHNOLOGY
Recombinant DNA and its Applications
Michael J. Freudiger 2008
References and Source Material:
Nelson, David L. and Michael M. Cox: Lehninger Principles of Biochemistry, 4th ed. W.H. Freeman & Co., 2005.
Lippert, Ross A., Introduction to Computational Molecular Biology, Lecture 2: Brute Force and Digestion Problems, Massachusetts Institute of Technology.
The Astbury Centre for Structural Molecular Biology
Michael J. Freudiger
and others, mod.

2. Dolly and surrogate Mom
Biotechnology
Dolly and surrogate Mom
Embryonic stem cells and gene therapy
Genetically modified rice.

3. Biotechnology
Biotechnology, defined broadly, is the engineering of organisms for useful purposes.
Often, biotechnology involves the creation of hybrid genes and their introduction into organisms in which some or all of the gene is not normally present.
Fourteen month-old genetically engineered (“biotech”) salmon (left) and standard salmon (right).

4. Biotechnology
The use of living cells to make products such as pharmaceuticals, foods, and beverages
The use of organisms such as bacteria to protect the environment
The use of DNA science for the production of products, diagnostics, and research

5. What Are Some Applications of Recombinant DNA Technology?
Bacteria, Yeasts, and Plants can all be modified to produce important pharmaceuticals, enriched foods, and industrial products.
Novalin-R (Insulin Regular) is a product of recombinant DNA technology, and helps diabetics to regulate blood sugar.
Synagis (Palivizumab) is a product of recombinant DNA technology and is given to babies who may be at risk of respiratory syncytial virus (RSV).
The flask and dish image is meant to illustrate yeasts modified with recombinant DNA to produce any number of products. The product is produced and then purified out of the growth media.
The corn cob is representative of the Genetically Modified Food Industry, where food crops have been modified to resist certain pests (including insects and fungi), or certain chemicals like Roundup (glyphosphate herbicide), and to reduce costs and improve yields of agricultural crops. 5

6. Gene cloning for pharmaceutical production
We’ll examine
Animal cloning
Genetically modified foods and the American-European opinion divide.
Gene cloning for pharmaceutical production
DNA fingerprinting
Gene therapy
The promise and perhaps perils of embryonic stem cells

7. 1 - Animal cloning
Dolly and her surrogate mother.

8. Why Clone Animals? To answer questions of basic biology
Five genetically identical cloned pigs.
To answer questions of basic biology
For pharmaceutical production.
For herd improvement.
To satisfy our desires (e.g. pet cloning).

9. Is Animal Cloning Ethical?
The first cloned horse and her surrogate mother/genetic twin.
As with many important questions, the answer is beyond the scope of science.

10. The Biotechnology of Reproductive Cloning
Even under the best of circumstances, the current technology of cloning is very inefficient.
Cloning provides the most direct demonstration that all cells of an individual share a common genetic blueprint.

11. Saved by Cloning?
Some are firm believers while many view these approaches to be more of a stunt.
Note the use of a closely related species, a domestic goat, as egg donor and surrogate mother.

12. The Next Step? Highly unlikely.
Attempts at human cloning are viewed very unfavorably in the scientific community.

13. 2 - Gene cloning for pharmaceutical production

14. Recombinant DNA
The manipulation and combination of DNA from two sources
Bacterial DNA + human gene for insulin
Plant DNA + bacterial DNA - Agrobacterium tumefaciens
Mouse DNA + human DNA = transgenic

15. Recombination Insert a foreign gene into a host
Plasmid ( for example, exogenous DNA) into the bacterial cell – transformation or transfection-organism referred to as transgenic ( eukaryote ) or recombinant( prokaryote)
Goal – To produce many copies ( clones) of a particular gene
Reporter gene – tags gene of interest – to identify the presence of a gene

16. Vectors
Plasmids
Viruses
Particles ( DNA coated bullets)
Exogenous DNA

17. Characteristics of a Vector
Can replicate independently in the host cell
Has restriction sites in the vector- cloning region
Has a reporter gene that will announce its presence in the host cell
Is a small size in comparison to the host chromosome for ease of isolation

18. Plasmids and restriction enzymes
What are Plasmids?
How can we modify plasmids?
Restriction Enzymes
Origins of restriction enzymes.
A close look at restriction enzymes.
Understanding plasmid diagrams.
This slide is meant to introduce the first part of the lecture. Every question proposed here will be answered shortly.

19. What are Plasmids?
In this Lecture…
Circular DNA that is used by bacteria to store their genetic information.
Modifying plasmids to include extra genes allows for the production of new proteins.
This slide is meant to introduce the first part of the lecture. Plasmids are circular DNA used by bacteria to store their genetic information. Modifying plasmids to include extra genes allows for the production of new proteins.

20. How Can We Modify Plasmids?
In this Lecture…
Restriction Enzymes
BamHI, HindIII, etc.
Where do they come from?
How do they work?
Different restriction enzymes do different things.
DNA Ligase
This slide is meant to introduce the first part of the lecture. Every question proposed here will be answered shortly.
Restriction Enzyme attached to DNA before cleavage

21. Origins of Restriction Enzymes
Bacteria produce restriction enzymes to protect against invading viral DNA/RNA.
More details can be given if necessary, however all the students need to know is that the bacteria have biologically designed the restriction enzymes as a defense mechanism. Many restriction enzymes exist, and they all do similar things, however their restriction sites (the site of DNA cleavage) differ in their base recognition sequences.

22. Origins of Restriction Enzymes
The enzymes cut the invading DNA/RNA, rendering it harmless.

23. Restriction Enzymes are Enzymes That Cut DNA Only at Particular Sequences
The enzyme EcoRI cutting DNA at its recognition sequence
Restriction enzyme animation
Different restriction enzymes have different recognition sequences.
This makes it possible to create a wide variety of different gene fragments.

24. Restriction Enzyme in Action
Sticky Ends
This slide shows a restriction enzyme cutting DNA. The enzyme used here is EcoRI, which cuts at any site where the base sequence is GAATTC.
DNA strand with EcoRI restriction site highlighted.
EcoRI restriction enzyme added (outline of separation about to occur).
Restriction fragments separate, with “sticky ends” at each edge.

25. Adding DNA Ligase Sticky Ends
DNA ligase bonds sticky ends cut with the same restriction enzyme.
Sticky ends cut with different restriction enzymes will not bond together.
Why?
Because the base pair sequence of the two sticky ends will be different and not match up.

26. DNAs Cut by a Restriction Enzyme Can be Joined Together in New Ways
These are recombinant DNAs and they often are made of DNAs from different organisms.

27. Plasmid Maps Indicate Restriction Sites and Genes
This slide is meant to show the complexities in mapping DNA. Notice the many mapped restriction sites and the 3 genes labeled on the plasmid. This plasmid is 2686 base pairs in size.

28. Make Recombinant DNA Using Restriction Enzymes
Recombinant DNA- Example
Make Recombinant DNA Using Restriction Enzymes
This next series of slides is meant to use the knowledge from the previous slides and simply make recombinant DNA.

29. DNA From Two Sources (Restriction Sites Labeled)
Here we are presented with the DNA to be cut. The restriction sites for a particular restriction enzyme have been labeled. Take note of the red area in the linear DNA. The goal of this series of slides is to produce a plasmid containing a single copy of this red sequence.
Circular DNA
Linear DNA

30. Application of Restriction Enzymes

31. Adding DNA Ligase
Adding DNA ligase will cause the “sticky ends” to come together, and many combinations are possible.

32. Recombinant DNA Plasmid
Many possible recombinant DNA plasmids can be produced, but this was the desired plasmid for the experiment.

33. Many Other Recombinant Possibilities
…and many more!

34. Plasmid DNA Insertion
DNA plasmids can be inserted into bacteria using a variety of laboratory processes.

35. Transgenic Colony Allowed to Grow

36. How Do We Get the Desired Plasmid?
Recombinant plasmids
Restriction fragments will ligate randomly, producing many plasmid forms.
Bacterial insertion would be necessary, then colony growth, and further testing to isolate bacteria with the desired plasmid.
Transformation of bacterial cells through electroporation.
Bacteria are then moved to a growth plate, and grown on selective media to “weed out” cells that have not picked up the desired plasmid.

37. Harnessing the Power of Recombinant DNA Technology – Human Insulin Production by Bacteria

38. Human Insulin Production by Bacteria
and cut with a restriction enzyme
6) join the plasmid and human fragment

39. Human Insulin Production by Bacteria
Mix the recombinant plasmid with bacteria.
Screening bacterial cells to learn which contain the human insulin gene is the hard part.

40. Route to the Production by Bacteria of Human Insulin
One cell with the recombinant plasmid
A fermentor used to grow recombinant bacteria.
This is the step when gene cloning takes place.
The single recombinant plasmid replicates within a cell.
Then the single cell with many recombinant plasmids produces trillions of like cells with recombinant plasmid – and the human insulin gene.

41. Route to the Production by Bacteria of Human Insulin
The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene.

42. Route to the Production by Bacteria of Human Insulin
Overview of gene cloning.
Cloning animation

43. Pharming
Pharming is the production of pharmaceuticals in animals engineered to contain a foreign, drug-producing gene.
These goats contain the human gene for a clot-dissolving protein that is produced in their milk.

44. Pharmaceuticals insulin for diabetics
factor VIII for males suffering from hemophilia A
factor IX for hemophilia B
human growth hormone (GH)
erythropoietin (EPO) for treating anemia
three types of interferons
several interleukins
granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone marrow after a bone marrow transplant
tissue plasminogen activator (TPA) for dissolving blood clots
adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID)
angiostatin and endostatin for trials as anti-cancer drugs
parathyroid hormone
hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B virus

45. 3 - Embryonic stem cells

46. The Promise and Possible Perils of Stem Cells

47. The Stem Cell Concept
A stem cell is an undifferentiated, dividing cell that gives rise to a daughter cell like itself and a daughter cell that becomes a specialized cell type.

48. Stem Cells are Found in the Adult, but the Most Promising Types of Stem Cells for Therapy are Embryonic Stem Cells

49. The Inner Cell Mass is the Source of Embryonic Stem Cells
The embryo is destroyed by separating it into individual cells for the collection of ICM cells.

50. Some Thorny Ethical Questions
Are these masses of cells a human?
Is it ethical to harvest embryonic stem cells from the “extra” embryos created during in vitro fertilization?

51. Additional Potential Dilemmas – Therapeutic Cloning to Obtain Matched Embryonic Stem Cells
Cultured mouse embryonic stem cells.
Cells from any source other than you or an identical twin present the problem of rejection.
If so, how can matched embryonic stem cells be obtained?
A cloned embryo of a person can be made, and embryonic stem cells harvested from these clones.

52. Therapeutic Cloning
Is there any ethical difference between therapeutic and reproductive cloning?

53. 4 - DNA, the Law, and Many Other Applications –
The Technology of DNA Fingerprinting
A DNA fingerprint used in a murder case.
The defendant stated that the blood on his clothing was his.
What are we looking at? How was it produced?

54. DNA Fingerprinting Basics
Different individuals carry different alleles.
Most alleles useful for DNA fingerprinting differ on the basis of the number of repetitive DNA sequences they contain.

55. DNA Fingerprinting Basics
If DNA is cut with a restriction enzyme that recognizes sites on either side of the region that varies, DNA fragments of different sizes will be produced.
A DNA fingerprint is made by analyzing the sizes of DNA fragments produced from a number of different sites in the genome that vary in length.
The more common the length variation at a particular site and the greater the number the sites analyzed, the more informative the fingerprint.

56. A Site With Three Alleles Useful for DNA Fingerprinting
DNA fragments of different size will be produced by a restriction enzyme that cuts at the points shown by the arrows.

57. The DNA Fragments Are Separated on the Basis of Size
The technique is gel electrophoresis.
The pattern of DNA bands is compared between each sample loaded on the gel.
Gel electrophoresis animation

58. Possible Patterns for a Single “Gene” With Three Alleles
In a standard DNA fingerprint, about a dozen sites are analyzed, with each site having many possible alleles.

59. A DNA Fingerprint
When many genes are analyzed, each with many different alleles, the chance that two patterns match by coincidence is vanishingly small.
DNA detective animation
HGP fingerprinting page

60. 5 - Genetically Modified Foods
Many of our crops in the US are genetically modified.
Should they be?

61. GM Crops are Here Today
Source: Pew Initiative on Food and Biotechnology, August 2004.

62. Methods for Plant Genetic Engineering are Well-Developed and Similar to Those for Animals

63. Golden Rice is Modified to be Provide a Dietary Source of Vitamin A
Golden rice (yellow) with standard rice (white).
Worldwide, 7% of children suffer vitamin A deficiency, many of them living in regions in which rice is a staple of the diet.

64. Genetically Modified Crops
Genetically Modified Cotton
(contains a bacterial gene for pest resistance)
Standard Cotton

65. GMOs, Especially Outside the US, Are a Divisive Issue
Protesters at the 2000 Montreal World Trade Summit
European sentiment

66. Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops
The jury’s still out on the magnitude of GM crop’s ecological impact, but the question is debated seriously.

67. Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops

68. 6 - Gene Therapy
Involves delivery of therapeutic genes into the human body to correct disease conditions created by faulty genes
Two primary strategies:
Ex vivo gene therapy
In vivo gene therapy

69. Ex vivo gene therapy Cells from diseased person are removed
Then, they are treated in lab (using techniques similar to bacterial transformation)
Finally, they are reintroduced to the patient
More effective than in vivo
Transfection is the introduction of DNA into animal or plant cells

70. In vivo gene therapy
Introducing genes directly into tissues or organs without removing body cells
Challenge is delivery only to intended tissues
Viruses act as vectors for gene delivery, but some injected directly into tissue

71. Delivery of therapeutic genes
Therapeutic genes often called payload
May require long-term expression of corrective gene
Others require rapid expression for short periods of time

72. Viral vectors
Viral vectors use viral genome to carry therapeutic gene(s) and to infect human body cells
Adenovirus (common cold)
Adeno-Associated Virus
Retrovirus (HIV)
Herpes simplex virus (cold sores)
Viruses must be engineered so that they can neither produce disease nor spread (extremely effective at infecting human cells)

73. Vector Transfection
Targeted gene therapy may result since some viruses infect certain body cells
Adenoviruses infect both dividing & non-dividing cells effectively
Adeno-Associated viruses do not cause illness in humans, can infect a wide variety of cells, & integrate 95% of time in same location
Retroviruses are of interest because they insert DNA into the genome of host where it remains permanently (integration), but often, randomly

74. Vector Transfection
Herpes virus (HSV-1) strain primarily affects central nervous system (CNS)
May help develop treatments for Alzheimer’s, Parkinson’s, and other genetic neurodegenerative diseases
However, although viral vectors may help, most human cells are not easily transfected

75. Alternative delivery
Liposomes are small diameter, hollow particles made of lipid molecules
Packaged with genes and injected into tissues
A gene gun could also be used
“Naked” DNA injected directly into body tissues (ex. effective in liver/muscle), but not enough cells express gene to have affect
Artificial chromosomes may also deliver therapeutic gene
Non-protein coding DNA with therapeutic gene
Similar construction to normal chromosomes (designed for permanent incorporation)

76. Curing Genetic Disease
More than 3,oo0 human genetic disease conditions are caused by single genes
These are candidates for treatment by gene therapy
Cystic Fibrosis
Huntington’s disease
Tay-Sachs
Hemophilia
Sickle cell disease
Phenylketonuria (PKU)

77. Classic Example: Cystic Fibrosis
Occurs with 2 defective copies of gene encoding the protein called cystic fibrosis transmembrane conductance (CFTR)
Normally it serves as a pump at the cell membrane to move chloride ions out of the cells
If cells can’t move chloride out, they absorb water trying to dilute the chloride in the cell
This leads to the production of THICK sticky mucus that clogs airways; ideal environment for infections (leading to pneumonia, etc…)

78. Treating Cystic Fibrosis
Back clapping
Drugs that thin mucus
Antibiotic treatment (to fight infections)
One form of gene therapy has helped

79. CF Gene Therapy Uses viruses and liposomes sprayed into nose & mouth
Expensive treatment
NOT a reliable cure yet
Requires multiple applications (DNA doesn’t integrate)
May not produce adequate protein

80. Unresolved Questions Can gene expression be controlled in the patient?
What happens if normal gene is overexpressed?
How long will the therapy last?
What is the best vector to use?
What is the minimum number of cells needed to infect to achieve success?