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Al-Walid Khalid Al-Shekri, OPI

Group 2020, {4th year}

Recombinant DNA Technology & Pharmaceutical Products

Contents :-

contents

  • 1-Introduction of biotechnology.

  • 2-Introduction of recombinant DNA technology (RDT).

  • 3-Techniques used in r-DNA technology.

  • 4-Gene cloning (r-DNA).

  • 5-Gene delivery.

  • 6-Gene therapy.

  • 7-Pharmaceutical products.

  • 8-Application of RDT.

  • 9-Conclusion.

  • references & questions

1-Introduction of biotechnology

1-Introduction of biotechnology

  • Bio-Techno-Ology.

  • 1-Bio -means life.
  • 2-Techno -means applying scientific knowledge for practical purposes.
  • 3-Ology –means the study of.

  • So, biotechnology: it means a study of how to apply technology to living systems.

  • Biotechnology involves the utilization of living organisms in industrial processes, particularly in 1-agriculture 2-food processing and 3-medication.

  • A-wine B-beer C-cheese D-bread

2-Introduction of recombinant DNA technology (RDT)

  • RDT also known as 1-gene cloning 2-molecular biotechnology 3-molecular cloning.

  • The ability to join a sequence of deoxyribonucleic acid (DNA) of interest to a vector that can then be introduced into a suitable host.

  • Stanley Cohen of Stanford University, Stanford, California.
  • Herbert Boyer at the University of California at San Francisco.

  • The first commercial production of recombinant human insulin.

  • More than 200 new drugs produced by recombinant DNA technology have been used to treat over 300 million people for diseases.

  • Over 400 new drugs are in the process of being tested in human trials to treat a variety of serious human diseases.

3-Techniques used in r-DNA technology

3-Techniques used in r-DNA technology

3-Techniques used in r-DNA technology

Gel electrophoresis

Gel electrophoresis

  • DNA fragments of different sizes can be separated by an electrical field applied to a “gel”.

  • The negatively charged DNA migrates away from the negative electrode and to the positive electrode.

  • The smaller the fragment the faster it migrates.

Restriction enzyme mapping

  • Frequently it is important to have a restriction enzyme site map of a cloned gene for further manipulations of the gene. This is accomplished by digestion of the gene singly with several enzymes and then in combinations. The fragments are subjected to gel electrophoresis to separate the fragments by size and the sites are deduced based on the sizes of the fragments.

Restriction enzyme mapping

Polymerase chain reaction (PCR)

  • It is an amplification technique for cloning the specific or targeted parts of a DNA sequence to generate thousands to millions of copies of DNA.

  • Allows the isolation of a specific segment of DNA from a small DNA (or cell sample) using DNA primers at the ends of the segment of interest.

Polymerase chain reaction (PCR)

1-The four deoxy-ribonucleotides.

2-Thermo-stable DNA polymerase (Taq polymerase).

3-Template sequence in a DNA sample.

4-Primers.

5- Buffer.

components

Steps

  • There are three steps of PCR :

1- Denaturation : heating to 95°c.

2- Annealing : cooling to 50 °c.

3- Extension : reheating to 72 °c.

Nucleic Acid Hybridization

  • A) Southern Blot allows the detection of a gene of interest by probing DNA fragments that have been separated by electrophoresis with a “labeled” probe.

  • B) Northern Blot (probe RNA on a gel with a DNA probe).

  • C) Western Blot (probe proteins on a gel with an antibody).

Nucleic Acid Hybridization

DNA Microarrays

  • DNA microarray is a collection of microscopic DNA spots attached to a solid surface.

  • DNA microarrays to measure the expression levels of large numbers of genes.

DNA Microarrays

steps of cloning

4-Gene cloning (r-DNA)

1-Obtain the target gene.

  • A-Isolation of target gene from organism. use restriction enzymes.

  • B-Preparation of complementary DNA (cDNA).

  • C-Chemical synthesis of DNA.

2-Finding suitable cloning vector.

  • A- Plasmid.

  • B- Bacteriophage.

  • C- Cosmid.

3- Join target gene to vector.

3- Join target gene to vector.

4- Transformation of r-DNA to Host cell

  • It is the process of introducing purified cloned DNA to a host cell. This process can be done by thermal shock or electrical shock or using chemicals as CaCl2. The cells must be prepared in a specific manner to receive the r-DNA.

4-Transformation of r-DNA to Host cell.

Selection Methods

  • 1- AB resistance marker: used to identify cells carry plasmid vector directly.

  • 2- Detect foreign DNA by detecting the desired gene product.

  • 3- Use a short piece of labeled DNA to identify cells carry the desired gene.

  • 4- Blue/ white screening.

Selection Methods

Gene delivery

5-Gene delivery

  • Gene delivery is the process of introducing foreign DNA into a host cell.

  • There a wide range of gene delivery developed for various types of cells and tissues, from bacterial to mammalian.

  • For gene delivery to be successful, foreign DNA must survive long enough in the host cell to integrate into a genome.

  • Genes are made of DNA. Effective gene delivery requires an efficient method to get the DNA into a cell and to make it work. So, DNA delivery "vehicle" as vectors.

  • There are no " perfect vectors" that can treat every disorder.

  • Part of the challenge in gene therapy is choosing the most suitable vector for treating the disorder.
  • To be successful, a vector must:-

  • 1- Target the right cells.

  • 2- Integrate the gene in the cells.

  • 3- Activate the gene.

  • 4- Avoid harmful side effects.

Cont

Cont

Vectors utilized as the method for gene delivery can be divided into two categories:-

1- Viral vector.

2- Non-viral.

Viral vectors

  • All viruses bind to their hosts and introduce their genetic material into the host cell as a major aspect of their replication cycle.
  • 1- They are very good at targeting and entering the cells.

  • 2- Some target specific types of cell.

  • 3- They can be modified so that they can't replicate and destroy cells.

advantages

disadvantages

  • 1- They can carry a limited amount of genetic material.

  • 2- They can cause immune responses.

  • 3- Expensive.

Examples

  • 1- Adenoviruses.

  • 2- Retroviruses.

  • 3- Lentiviruses.

  • 4- Herpes simplex viruses.

Non-viral vectors

plasmid

  • Non-viral vectors, mainly plasmids, play a major role in transferring genes into host cells for protein expression. Plasmids as carriers have been involved in many published successful results. The most important advantages of using plasmids instead of viruses for protein production are biosafety for operators and product safety for patients. The safety requirements for large scale manufacturing of plasmid DNA (one gram of plasmid DNA is suitable for 1000 L transfection) are much lower than those required for working with a large quantity (>1014 infectious units) of potentially infectious virus. The entire production process of plasmid preparation, transfection, product harvest, and purification can be managed in a regular production environment as for other recombinant proteins. The advantages of using plasmids for TGE also include ease of construction, amplification, and purification of plasmids. Delivery of the plasmid into cells is simple via inexpensive chemical agents. The disadvantages are lower transfection efficiency (20-80% with some chemical reagents) than viral vectors as well as a size limitation that usually allows only two genes on a single plasmid.

liposomes

  • They are empty circles surrounded by a lipid bilayer.

  • They can be filled with DNA or proteins such as TNF.

  • Liposomes interact with cell membranes because both have hydrophobic lipid layers.

  • Once in contact, the liposome converges with the cell membrane, delivering its payload into the cell.

liposomes

6-Gene therapy

  • Gene therapy is the utilize of genes as a drug.

  • The main focus of gene therapy is the replacement of faulty genes with healthy genes.

  • In some cases, the healthy genes are utilized to fix or turn-off the faulty genes.

  • Introducing a single good copy of the gene can then cure the defect (sometimes known as replacement gene therapy).

  • Aggressive(suicide) gene therapy uses genes to kill or destroy unwanted cells and is especially useful against cancer.

Pharmaceutical products

  • A foreign gene is replicated and expressed in the bacteria. Thus a large amount of protein product is obtained. These products are now available in markets.

7-Pharmaceutical products

Insulin

  • Pancreatic beta cells.

  • It enables cells to take up glucose from the bloodstream to use in the production of ATP.

  • Insufficient insulin causes diabetes.

  • Before recombinant insulin was available, insulin was obtained from cows’ or pigs’ pancreases.

Human Growth Hormone (HGH)

Human growth hormone (HGH)

  • Also know as somatotropin

  • Promotes body growth by increasing amino acid uptake by cells, protein synthesis and fat utilization for energy.

  • Dwarfism(caused by insufficient production of HGH by the pituitary gland).

  • Production of recombinant HGH

Old method

Old method

  • Purification of HGH from cadaver pituitary glands.

• 8 cadavers/year for 8 – 10 years per patient

New method

Gene production for HGH synthesis

• Protropin Genentech.

• Humatrope Eli Lilly.

Tissue plasminogen activator (tPA)

  • It is a serine protease found on endothelial cells.

  • Breakdown of blood clots (fibrinolysis).

  • tPA enzyme catalyzes the conversion of plasminogen to plasmin.

  • Treat coronary thrombosis.

  • Recombinant tissue plasminogen activator (r-tPA).

produce tPA by r-DNA technology

  • a. Streptokinase once used for this purpose:-

– Derived from Streptococcal bacteria.

– Must be delivered to blood vessel directly.

  • b. Urokinase = alternative, but has a risk of hemorrhage.

produce tPA by r-DNA technology

tPA By Genentech

  • Activase (Alteplase recombinant) is the trade name of Genentech’s t-PA. Activase is useful in treating heart attacks and strokes when administered within 5 hours of thrombosis formation or embolism lodging in the heart or brain.

  • FDA approves “Activase” – Genentech – 1987

– Clot-Dissolving Agent

– Fewer side effects than streptokinase and urokinase.

tPA By Genentech

Hemophilia factor VIII

Hemophilia factor VIII

  • Since the early 1990s, recombinant human clotting factor VIII (r-hFVIII) produced in hamster cells.

  • Chinese hamster ovary (CHO) or baby hamster kidney cells (BHK).

  • Factor VIII is an essential blood-coagulating protein, (called anti-hemophilic factor (AHF)).

  • cDNA obtained from gene sequence and cloned into Hamster Kidney Cells to produce Factor VIII protein. However, E. coli not used because protein needs extensive glycosylation.

  • Recombinate & Kogenate.

Vaccine

Recombinant vaccine

  • A vaccine is a preparation of killed or weakened microorganism that is given to a person orally or injected in order to prevent disease.

  • Edward Jenner in 1798 used cowpox virus to immunize people against smallpox.

  • Properties of good vaccine: 1-safety 2-stability 3-inexpensive 4-long term protection.

  • Types of vaccines: 1-live vaccine 2-killed vaccine 3-recombinant vaccine.

  • The genes encoding any immunogenic proteins can be cloned and expressed in bacterial, yeast and mammalian cells using r-DNA technology.

  • The first such recombinant antigen vaccine approved for human use is hepatitis B vaccine.

  • This vaccine was developed by cloning the gene for the major surface antigen of hepatitis B virus (HBsAg) and expressing it in yeast cells.

Recombinant vaccine

The recombinant vaccines may be broadly categorized into three groups:

  • 1. Subunit recombinant vaccines: These are the components of the pathogenic organisms. Subunit vaccines include proteins, peptides and DNA.

  • 2. Vector recombinant vaccines: These are the genetically modified viral vectors that can be used as vaccines against certain pathogens.

  • 3. Attenuated recombinant vaccines: These are the genetically modified pathogenic organisms (bacteria or viruses) that are made non-pathogenic and used as vaccines.

Recombinant vaccine

Advantages

  • Produced more quickly. In larger quantities.

  • Free from infectious virus particles.

  • They can safely be given to immuno-suppressed people

  • They are less likely to induce side effects.

Advantages

Disadvantages

  • Antigens may not retain their native conformation, so that antibodies produced against the subunit may not recognize the same protein on the pathogen surface.

  • Isolated protein does not stimulate the immune system as well as a whole organism vaccine.

Monoclonal anti-body

  • An antibody is a protein produced by the body's immune system in response to antigens, which are harmful substances.

  • Antigens include bacteria, fungi, parasites, viruses, and chemicals, which the immune system identifies as foreign.

  • scientists can produce antibodies in the lab that mimic the action of the immune system.

  • The mice are vaccinated with the human antigen that scientists want to produce antibodies against. (This causes the immune cells of the mice to produce the desired human antibody).

  • The term monoclonal antibody means that the man-made antibody is synthesized from cloned immune cells, and the identical monoclonal antibody produced binds to one type of antigen. (what different between monoclonal & Polyclonal antibodies??)see the poster

production of monoclonal anti-body

Interferon

  • Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of pathogens, such as viruses, bacteria, parasites, or tumor cells.

  • a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.

Interferon

Production of Recombinant Interferons

  • Recombinant DNA technology has proved the most satisfactory route to the large scale production of human interferons.

  • The genes of all three types of HuIFN have been cloned in micro-organisms and expression obtained.

  • HuIFN β and γ produced in this manner lack the glycosylation present in the naturally occurring substances but this does not affect their specific activity.

  • Greatly improved methods of purification, including immuno-adsorption chromatography on monoclonal antibody columns, are now available so there should be no difficulty in supplying adequate amounts of very pure interferon of all three types although, up till now, only HuIFN α has been readily available.

Production of Recombinant Interferons

Erythropoietin (EPO)

  • Human Erythropoietin is produced in the kidney.

  • It is a glycoprotein, acts on the bone marrow to increase the production of red and white blood cells.

  • Widely used in AIDS for development of immunity.

Erythropoietin (EPO)

Production of recombinant EPO

  • Isolating and constructing human EPO cDNAs.

  • Subjecting the cDNA to PCR using primers based on the published sequence.

  • The PCR products will be cloned into a vector for the purpose of propagation and subsequently engineered into appropriate expression vectors.

  • Culture- production-purification.

Production of recombinant EPO

Example of EPO

  • a. Epoetin-alfa “Epogen”:- treatment of anemia due to chronic renal failure.

  • c. Filgrastim“Neupogen”:- Stimulates stem cells to produce neutrophils (and other leukocytes).

Example of EPO

Application of r-DNA technology

8-Application of RDT

Production of Transgenic Plants

  • Also known as genetically modified plants.

  • Transgenic plants have been developed with better qualities like resistance to herbicides, insects or viruses or with the expression of male sterility, etc.

  • Promoting health in plants

Transgenic Plants

  • Transgenic bacteria are used to promote the health of plants.

  • A bacterium normally forms colonies in the roots of corn plants.

  • These genes code for an insect toxin. These toxins protect the root from the insect.

Promoting health in plants

Production of Transgenic Animals

  • By the use of r-DNA technology, desired genes can be inserted into the animal so as to produce the transgenic animal.

  • Aids the animal breeders to increase the speed and range of selective breeding in case of animals.

  • Use of transgenic animals is the production of certain proteins and pharmaceutical compounds.

  • Biotechnologists have successfully produced transgenic pigs, sheep, rats, and cattle.

Transgenic Animals

Production of Antibiotics

Antibiotics

  • Antibiotics produced by microorganisms are very effective against different viral, bacterial or protozoan diseases.

  • Some important antibiotics are tetracycline, penicillin, streptomycin, novobiocin, bacitracin, etc.

  • r-DNA technology helps in increasing the production of antibiotics by improving the microbial strains through modification of genetic characteristics.

Production of Commercially Important Chemicals

Important Chemicals

  • Various commercially important chemicals can be produced more efficiently by utilizing the methods of r-DNA technology.

  • A few of chemical are the alcohols and alcoholic beverages obtained through fermentation;

  • Organic acids like citric acid, acetic acid, etc. and vitamins produced by microorganisms.

Prevention and Diagnosis of Diseases

Prevention and Diagnosis of Diseases

  • For diagnosis of disease.

  • For prediction various mutations.

  • For identification of the people who have a family history including recessive genetic disorders.

  • For prenatal diagnosis by testing a fetus when he has a risk of being a child with mental or physical disabilities.

  • As a preventative measure once the baby is born.

Applications in forensic science

Applications in forensic science

  • The applications of r-DNA technology in forensic sciences largely depend on the technique called DNA profiling or DNA fingerprinting.

  • It enables us to identify any person by analyzing his hair roots Wood stains, serum, etc.

  • DNA fingerprinting also helps to solve the problems of parentage and to identify the criminals.

Biofuel Production

  • Biofuels are derived from biomass and these are renewable and cost-effective.

  • r-DNA plays an essentially important role in beneficial and large scale production of biofuels like biogas.

  • Bio-hydrogen, bio-diesel, bio-ethanol..., etc.

  • Genetic engineering helps to improve organisms for obtaining higher product yields and product tolerance.

Biofuel Production

10-Conclusion

9-Conclusion

  • r-DNA technology can be treated several diseases which cannot treat before such as some types of cancer, hemophilia, and dwarfism. Also, r-DNA technology has prevented the human from many serious diseases by making a vaccine.

  • This just the beginning.

  • recombinant DNA technology has a large scale of utilization in the future.

  • The challenges in improving the products at gene level sometimes face serious difficulties which are needed to be dealt with for the betterment of the recombinant DNA technology future.

  • r-Vaccines are in early phase. So, r-Vaccines are going to be the vaccines of next generation.

  • does r-DNA technology make the world free of disease?

references

References

[1]:- Cummings R. (2019) Biotechnology. 1sted. USA: Yashon R ; p.1-2.

[2]:- hegazy, w. (2017). Biotechnology. 1st ed. muscut: Ministry of health, p.2.

[3]:- R.Glick, B., L.Delovitch, T. and L.Patten, C. (2014). Medical Biotechnology. 1st ed. Washington: American Society for Microbiology, p.3.

[4]:- R.Glick, B., Pasternak, J. and L.Patten, c. (2010). Molecular Biotechnology principles & application of recombinant DNA. 4th ed. Washington: American Society for Microbiology, pp.108 - 110.

[5]:- T.A., B. (2010). Gene Cloning and DNA Analysis. 6th ed. UK, pp.5 - 8.

[6]:- zhu, j. (2019). update on production of recombinant therapeutic proteins transient gene expression. 1st ed. Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK: Smithers Group Company, p.23.

[7]:- P.clark, D. and J.Pazdernik, N. (2012). biotechnology. 1st ed. California 92101-4495, USA: Elsevier, p.488.

[8]:- Khan, S., Wajid Ullah, M. and Siddique, R. (2016). Role of Recombinant DNA Technology to Improve Life. [online] NCBI. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5178364/#sec5title [Accessed 20 Sep. 2019].

[9]:- T.A., B. (2010). Gene Cloning and DNA Analysis. 6th ed. UK, pp.252.

[10]:- T.A., B. (2010). Gene Cloning and DNA Analysis. 6th ed. UK, pp.253 - 254.

[11]:- R.Glick, B., Pasternak, J. and L.Patten, c. (2010). Molecular Biotechnology principles & application of recombinant DNA. 4th ed. Washington: American Society for Microbiology, pp.399.

[12]:- R.Glick, B., Pasternak, J. and L.Patten, c. (2010). Molecular Biotechnology principles & application of recombinant DNA. 4th ed. Washington: American Society for Microbiology, pp.403 -406.

[13]:- Griffiths AJF, Gelbart WM, Miller JH, et al. (1999) Modern Genetic Analysis New York:W. H. Freeman.UK

[14]:- S.S.SandhuRecombinant DNA technology I. K. International Pvt Ltd, 01-Jun-2010

thank you for your attention

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