Chapter 1: Introduction to Biotechnology PDF

3/30/2023 Chapter 1: Introduction to Biotechnology Pr. Said El Shamieh B3212 Molecular Biotechnology Bachelor of Biology...

3/30/2023 Chapter 1: Introduction to Biotechnology Pr. Said El Shamieh B3212 Molecular Biotechnology Bachelor of Biology 1 I.1 Biotechnology "Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use." Modern use of the term biotechnology includes genetic engineering as well as cell/ tissue culture technologies. Dr. Said El Shamieh 2 1  3/30/2023 Biotechnology is needed because: Nature has a rich source of variation Here we see bean has many seedcoat colors and patterns in nature But we know nature does not have all of the traits we need Dr. Said El Shamieh 3 II. History of Biotechnology For thousands of years, humans have used selective breeding to improve production of crops and livestock to use them for food. In China, soybean curds were used as antibiotics to treat boils in 500 BC, and, powdered chrysanthemum was used as insecticide in 100 BC. In Greece, crop rotation to maximize soil fertility was practiced in 250 BC. Egypt, and India developed the process of brewing beer. Other cultures produced the process of lactic acid fermentation which allowed the fermentation and preservation of other forms of food (dairy products). Fermentation was also used in this time period to produce leavened bread. Dr. Said El Shamieh 4 2  3/30/2023 II.2. Modern Biotechnology and the first GMOs In 1917, Chaim Weizmann was the first to use a pure microbiological culture in an industrial process, that of manufacturing corn starch using Clostridium acetobutylicum, to produce acetone, which the United Kingdom desperately needed to manufacture explosives during WWI. Dr. Said El Shamieh 5 In 1928, Alexander Fleming discovered the mold Penicillium and his work led to the discovery and purification of the antibiotic penicillin. In 1940, penicillin became available for medicinal use to treat bacterial infections in humans. Dr. Said El Shamieh 6 3  3/30/2023 Molecular Biotechnology Deals with The general principle of producing a GMO by add new genetic material into an organism's genome. This is called genetic engineering. KO Transgenic animals Dr. Said El Shamieh 7 Genetic engineering Dr. Said El Shamieh 8 4  3/30/2023 Some milestones in Molecular Biotechnology field A genetically modified micro-organism could be patented: Indian-born Ananda Chakrabarty, who was working for General Electric, had developed a bacterium derived from the Pseudomonas genus and capable of producing enzymes that break down crude oil, which he proposed to use in treating oil spills. Dr. Said El Shamieh 9 Milestones in Molecular Biotechnology In 1978 the company, Genentech founded by Herbert Boyer, announced the creation of an E. coli strain producing the human protein insulin: Using recombinant DNA technology, an existing bacterium. Genentech was the first company to use recombinant DNA technology. A genetically modified microorganism could be patented: Indian-born Ananda Chakrabarty, who was working for General Electric, had developed a bacterium derived from the Pseudomonas genus and capable of producing enzymes that break down crude oil, which he proposed to use in treating oil spills. Dr. Said El Shamieh 10 5  3/30/2023 Bacteria particularly important in producing large amounts of pure human proteins for use in medicine: Blood-clotting factors to treat haemophilia Human growth hormone to treat various forms of dwarfism. Anti-viral protein called interferon, the immune stimulant called interleukin 2, a tissue plasminogen activator for dissolving blood clots, and many other products. Dr. Said El Shamieh 11 As a result, nowadays there exist four main groups in biotechnological applications, which have been identified by a color system. THE COLORS OF BIOTECHNOLOGY Dr. Said El Shamieh 12 6  3/30/2023 Red biotechnology Brings together all those biotechnology uses connected to medicine. Includes producing vaccines and antibiotics, developing new drugs, molecular diagnostics techniques, regenerative therapies and the development of genetic engineering to cure diseases through genetic manipulation. Some relevant examples of red biotechnology are cell therapy and regenerative medicine, gene therapy and medicines based on biological molecules such as therapeutic antibodies. Dr. Said El Shamieh 13 White biotechnology All the biotechnology uses related to industrial processes - that is why it is also called ‘industrial biotechnology’ Pays a special attention to design low resource- consuming processes and products, making them more energy efficient and less polluting than traditional ones. Dr. Said El Shamieh 14 7  3/30/2023 Products issued from white biotechnology Dr. Said El Shamieh 15 Green biotechnology Focuses on agriculture as working field. It includes creating new plant varieties of agricultural interest, producing biofertilizers and biopesticides, using in vitro cultivation and cloning plants. Producing modified plant varieties is based almost exclusively on transgenesis, or introducing genes of interest from another variety or organism into the plant. Dr. Said El Shamieh 16 8  3/30/2023 Green biotechnology Three main objectives are pursued by using this technology: 1. Produce plant varieties resistant to pests and diseases. e.g: currently used and marketed maize varieties resistant to pests. 2. Developing varieties with improved nutritional properties e.g: higher content of vitamins. 3. Develop plant varieties able to act as bio-factories and produce substances of medical, biomedical or industrial interest in quantities easy to be isolated and purified. Dr. Said El Shamieh 17 Blue biotechnology Is based on the exploitation of sea resources to create products and applications of industrial interest. Taking into account that the sea presents the greatest biodiversity, there is potentially a huge range of sectors to benefit from the use of this kind of biotechnology. Dr. Said El Shamieh 18 9  3/30/2023 Industrial Products One of the latest amazing aquatic discoveries is directly related to marine biotechnology-that of thermophiles and psychrophiles. These are organisms that are capable of living in extreme environments. Enzymes produced by thermophiles have great importance in the industry of biotechnology. Those enzymes are the basis of the PCR. Psychrophile Grand Prismatic Spring, Yellowstone National Park 19 Dr. Said El Shamieh Chapter 2: Fundamental aspects of molecular Biotechnology Pr. Said El Shamieh B3212 Molecular Biotechnology Bachelor of Biology 10  3/30/2023 I.1 Recombinant DNA technology Is the creation of a new combination of DNA segments that are not found together naturally. RDT uses a group of, restriction endonucleases (RE), which cut the long strands of DNA at specific sites. These RE enzymes are made from bacteria and allow gene cloning by inserting the gene of interest into a vector, often a self-replicating bacterial which is a small circular DNA molecule with its own origin of replication. – Cloning vectors – Expression vectors Restriction enzymes allow cleavage of DNA into small fragments 22 11  3/30/2023 Restriction enzymes can generate sticky or blunt ends Figure 12.3b: Symmetrical cuts made in each Figure 12.3a: Asymmetrical cuts made by strand at the center of symmetry of the restriction enzymes in each strand at an equal restriction site. distance from the center of symmetry of the 23 restriction site DNA ligases allow insertion of DNA fragments into cloning vectors 24 12  3/30/2023 E.Coli plasmid vectors are suitable for cloning isolated DNA fragments Basic components of a plasmid cloning vector that can replicate within an E.coli cell25 Size Properties of vectors 13  3/30/2023 DNA cloning in a plasmid vector permits amplification of a DNA fragment 27 cDNAs prepared by reverse transcription of cellular mRNAs can be cloned to generate cDNA libraries 28 14  3/30/2023 cDNAs prepared by reverse transcription of cellular mRNAs can be cloned to generate cDNA libraries 29 cDNAs prepared by reverse transcription of cellular mRNAs can be cloned to generate cDNA libraries 30 15  3/30/2023 Phage cDNA libraries can be screened with a radiolabeled probe to identify a clone of interest 31 E.Coli expression systems can produce large quantities of proteins from cloned genes Dr. Said El Shamieh 32 16  3/30/2023 Plasmid expression vectors can be designed for use in animal cells Dr. Said El Shamieh 33 Plasmid expression vectors can be designed for use in animal cells Dr. Said El Shamieh 34 17  3/30/2023 II. The biological systems in prokaryotes and eukaryotes A. Prokaryotic cells lack a membrane-bound nucleus. Their DNA is naked within the cytoplasm in a region called nucleoid. Prokaryotes reproduce by means of binary fission, duplicating their genetic material and then essentially splitting to form two daughter cells identical to the parent. B. Eukaryotic cells have a nucleus. They also contain internal membrane-bound structures called organelles. Organelles, such as mitochondria and chloroplasts, are both believed to have evolved from prokaryotes that began living symbiotically within eukaryotic cells. Eukaryotic cells can reproduce in one of several ways, including meiosis and mitosis. There are two types of cultures: Microbiological cultures: for growing microorganisms such as bacteria or yeast Cell culture: for growing specific cell types derived from plants or animals. 18  3/30/2023 II.A.1. E. coli: Gram-negative, facultative anaerobic and non- sporulating bacterium that is commonly found in the lower intestine of warm-blooded organisms. Typical characteristics of E. coli cells include the following: Rod-shaped Optimal growth occurs at 37°C, but some laboratory strains can multiply at temperatures of up to 49°C. II.A.1. E. coli: Growth can be driven by aerobic or anaerobic respiration. Frequently used as a model organism in microbiological studies: for example to study conjugation. Ability to transfer DNA via bacterial conjugation, transduction or transformation. Very versatile host for the production of heterologous or recombinant proteins, allowing for the mass production of proteins in industrial fermentation processes. 19  3/30/2023 II.A.1. E. coli: Cons: E. coli cannot be used to produce some of the more large, complex proteins which contain multiple disulfide bonds and, or proteins that also require post-translational modification for activity. Another disadvantage is that E. coli does not secrete proteins well. II.A.2- Saccharomyces cerevisiae: 20  3/30/2023 II.A.2- Saccharomyces cerevisiae: It is a species of budding yeast. The most useful in the most common type of fermentation such as baking and brewing. S. cerevisiae cells are round to ovoid, Valuable for studies of other organisms including: the use of the yeast two-hybrid screening system and of YACs for cloning large fragments of DNA, II.A.2- Saccharomyces cerevisiae: One of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like E. coli as the model prokaryote. Is a single celled eukaryotic organism, small, with a short generation time (doubling time 1.5–2 hours at 30 °C) and can be easily cultured. In contrast to most other organisms, integrative recombination proceeds exclusively via homologous recombination. 21  3/30/2023 II.A.2- Saccharomyces cerevisiae: Unlike most other microorganisms, strains of S. cerevisiae have both a stable haploid and diploid state. Low percentage of non-coding DNA that can confound research in higher eukaryotes. S. cerevisiae was the first eukaryotic organism whose genome was completely sequenced (in 1996). Its genome is composed of about 12,156,677 base pairs and 6,275 genes, compactly organized on 16 chromosomes. Shares about 23% of its genome with that of humans. II.B. Eukaryotic cell cultures: Purpose: Two types of cells are cultured: a) Primary cells: cells that are cultured directly from a subject. b) Secondary / Immortalized cell line: a cell line that has acquired the ability to proliferate indefinitely. 22  3/30/2023 Stages in the establishment of a cell culture 45 II.B. Eukaryotic cell cultures: II.B.1. Isolation of cells to be cultured: (i) From blood (ii) From soft tissues: mononuclear cells can be released by enzymatic digestion with enzymes such as collagenase. (iii) Alternatively, pieces of tissue II.B.2- Maintaining eukaryotic cells in culture: Nutrients, Vitamins, a.a Growth Factors CO2, O2 Anti bacterial/ fungal agents.. 23  3/30/2023 II.B.3. Manipulation of cultured cells: Among the common manipulations carried out on cultured cells are: Media changes, Passaging of cells, and Transfecting cells: Introduction of foreign DNA into cells. DNA can also be inserted into cells using viruses. Anti-bodies production PRODUCTION OF HYBRIDOMA CELLS 24  3/30/2023 HAT medium is commonly used to isolate hybrid cells De novo and salvage pathways for nucleotide synthesis 49 Hybrid cells called hybridomas produce abundant monoclonal antibodies 50 25  3/30/2023 Protein sorting is the mechanism by which a cell transports proteins to the appropriate positions in the cell or outside of it. This delivery process is carried out based on information contained in the protein itself. SECRETION PATHWAYS Different types of signals 1. Signal Sequence: A peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. 2. The mitochondrial targeting signal (MTC) 3. The nuclear localization signal (NLS) 4. The peroxisomal targeting signals (PTS): These signal peptides directing to the. 26  3/30/2023 II.C.1. Secretion in eukaryotic cells 1. Post-translational translocation: Proteins destined for the mitochondria, chloroplasts, peroxisomes or nucleus. The proteins are translated on free ribosomes in the cytosol and later transported to their destination due to the specific sequences. II.C.1. Secretion in eukaryotic cells 2. Co-translational translocation: 1- The rough RE 2- The Golgi Glycosylation Activation 3- The cell membrane 27  3/30/2023 Depending on the cell type, there are two types of secretions: Constitutive secretion where proteins are transported directly to the plasma membrane via secretory vesicles Regulated secretion where more modification can occur to the proteins transported in the secretory granules; (insulin). 28  3/30/2023 III. How can genetically modified organisms (OGMs) be constructed? A genetically modified organism (GMO) is an organism whose genetic material has been altered using genetic engineering techniques. These techniques use recombinant DNA technology, thus transferring new DNA into an organism giving it modified or novel genes. Genetic modification involves the insertion or deletion of genes. => KO and trasgenic organisms Transgenic bacteria 29  3/30/2023 KO MOUSE Specific genes can be permanently inactivated in the germ line of mice 60 30  3/30/2023 Specific genes can be permanently inactivated in the germ line of mice 61 ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice 62 31  3/30/2023 ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice 63 ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice 64 32  3/30/2023 Somatic cell recombination can inactivate genes in specific tissues 65 I.1. DNA microinjection: 33  3/30/2023 Transgenic Plants Agrobacterium tumefaciens introduces a circular DNA molecule, called the Ti (tumor-inducing) plasmid, into the plant cell in a manner similar to bacterial conjugation. The plasmid DNA then recombines with the plant DNA. For making transgenic plants, desired foreign genes are cloned into the Ti plasmid vector and then introduced into plant cells via the Agrobacterium which infects and introduces the Ti plasmid into the host plant. Chapter 3: Manipulation of Gene Expression in Prokaryotes and Eukaryotes Pr. Said El Shamieh B3212 Molecular Biotechnology Bachelor of Biology 34  3/30/2023 I. Types of promoters used to regulate gene expression Regulation of gene expression is essential as it : Increases the versatility and adaptability by allowing the cell to express proteins when needed. Drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. Promoters used in biotechnology can be generally divided into: a- Constitutive promoters: These promoters are expressed in virtually all tissues and are largely, if not entirely, independent of environmental and developmental factors. Constitutive promoters are usually active across species and even across kingdoms. include actin, ubiquitin etc. 35  3/30/2023 b- Tissue-specific or development-stage- specific promoters: These promoters direct the expression of a gene in specific tissue(s) or at certain stages of development. Tissue specific promoters include root-specific, fruit specific, muscle specific etc. c- Inducible promoters: Their performance is conditioned by environmental conditions and external stimuli that can be artificially controlled. Promoters modulated by abiotic factors such as light, oxygen levels, heat (for ex. heat-shock promoter), cold and wounding. 36  3/30/2023 d- Synthetic promoters: Constructed by bringing together the primary elements of a promoter region from diverse origins. It is possible to synthesize consensus sequences that may work across different organisms and are not necessarily derived from a particular organism. II. Manipulation of gene expression in prokaryotes Operon specific units The ara operon encodes enzymes needed for the catabolism of arabinose to xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. It has both positive and negative regulation. 37  3/30/2023 II.A. Promoters used to drive transcription in prokaryotes (E. coli) 1- The lac promoter: It is inducible, regulated positively by lactose (or IPTG) and negatively by glucose. Modified lac promoters include: tac promoter which has the -10 sequence of lac and the -35 sequence of trp separated by 16 bp trc promoter which has the -10 of lac and the -35 of trp separated by 17 bp; trc and tac promoters are 3X stronger than trp and 10X stronger than lac. 2- The phage promoters: The lambda pL (or leftward) is a very strong promoter when active Completely shut down by the lambda repressor cI. 38  3/30/2023 Phage T7 promoter is a strong promoter and requires T7 RNAP for transcription; the T7 RNAP production can be placed under lac repressor control (lacIq preferred for tight control); high level of T7 RNAP expression can then be induced by IPTG and in its turn high level of T7 RNAP production would allow high target gene expression. 3- Broad host range promoters: E. coli is not always “best”. Other host strains may be preferred for producing certain products. Broad host range promoters have become useful in allowing expression in many strains/species – Cons: may not give maximum expression in all species. 39  3/30/2023 4- Promoters for Lactococcus species: Lactococcus species are commonly used in food production (cheese, yogurt, etc.) Knowing that the sequence between -10 and -35 promoter regions can affect the level of promoter activity Scientists synthesized Lactococcus promoters with random nucleotides between –10 and –35 and tested them for activity. Functional promoters with 400-fold range of strength were identified II.B. Prokaryotic expression vectors, protein folding, increasing protein secretion etc. 1- Prokaryotic expression vectors: In addition to basic components of a cloning vector (polylinker, ori and selectable marker) Inducible promoter placed upstream of the cloned sequence Transcription termination signal placed downstream of the cloned sequence A ribosome binding site (Shine-Dalgano sequence) placed downstream of the transcription start site. 40  3/30/2023 2- Protein folding and stability: Inhibition of post translational modifications: In some cases, host proteases may selectively cleave precursor forms of expressed proteins to produce mature active forms, which may not be desirable. Two common mutations in E. coli to eliminate the action of host proteases include the OmpT- and Lon- strains; OmpT and Lon genes code for proteases which can degrade expressed proteins. 2- Protein stability: Protein Tagging: Expressed proteins may be protected from degradation by adding a tag on the expressed protein. His-tags (6 Histidines): An affinity tag but does not increase the expression of the fusion protein or enhance its solubility. Purification from lysed cells is done under non-denaturing conditions by immobilization of His-fusion proteins onto nickel or cobalt-coated resin, which bind to the imidazole side chain of Histidine. Purification of denatured His-fusion proteins is done by lowering the pH (~6 for cobalt and ~4 for nickel); at high pH, histidine binds to nickel or cobalt. 41  3/30/2023 GST GSH-tag: Increases the expression and solubility of fusion proteins. Purification from lysed cells is done under non-denaturing conditions by absorption onto glutathione S Transferase-agarose beads, followed by elution in the presence of free glutathione. 42  3/30/2023 III. Manipulation of gene expression in eukaryotes Many handicaps are encountered when using E. coli to produce eukaryotic products: Protein instability Absence of post-translational protein modification such as glycosylation, Eukaryotic organisms have been studied as suitable replacements. Three types of eukaryotic hosts are generally used: Yeast, Insect cells Mammalian cells. III.1. The yeast expression systems, Saccharomyces cerevisiae A) Why yeast is especially suitable as a host: Easy to culture and their genetics and physiology have been well characterized. Carry out many post-translational modifications and expressed proteins are easily purified following secretion. Have an endogenous plasmid called the 2u plasmid which can be used as a cloning vector. DNA can be introduced to yeast by a variety of methods: some involve removal of the cell wall to form protoplasts, or in another method, called electroporation, cells are treated with pulses of electric current. 43  3/30/2023 B) The yeast expression vectors: Yeast expression shuttle vectors harboring a selectable marker have been used in the yeast system. Autotroph: Any organism that can synthesize its food from inorganic substances, using heat or light as a source of energy. Auxotroph: Organism that carries a mutation rendering it unable to synthesize an essential compound for its growth. Leu- 44  3/30/2023 Shuttle vectors and Eukaryotic cells Shuttle vectors include plasmids that can propagate in eukaryotes and prokaryotes: – e.g. both Escherichia coli and Saccharomyces cerevisiae Or in different species of bacteria – e.g. both E. coli and Rhodococcus erythropolis. The main advantage of these vectors is they can be manipulated in E. coli, then used in a system which is more difficult or slower to use (e.g. yeast). Yeast genomic libraries can be constructed with shuttle vectors 90 45  3/30/2023 Four types of shuttle vectors 1) Integrative plasmids (YIp): Integrated by homologous recombination into the host genome. Opened by restriction and linearized DNA is used for transformation; => this (normally) results in the presence of one copy of the foreign DNA inserted at this particular site. Four types of shuttle vectors 2) Episomal plasmids (YEp): They carry part of the 2 μ plasmid DNA sequence necessary for autonomous replication; Multiple copies of the transformed plasmid are propagated in the yeast cell and maintained as episomes. Episomal vectors give the highest levels of expression but tend to be unstable in large cultures. 46  3/30/2023 Four types of shuttle vectors 3) Autonomously replicating plasmids (YRp): They carry a yeast origin of replication (ARS sequence) that allows the transformed plasmids to be propagated several hundred-fold. This YRp plasmids is a Yip + ARS. It is a shuttle vector that can replicate in both yeast and E-coli; recombinant DNA is selected in bacteria using antibiotics, and yeast transformants are selected using media without tryptophan. Four types of shuttle vectors 4) Cen plasmids (YCp): ARS sequence, Centromeric sequence which normally guarantees stable mitotic segregation and reduces the copy number of self-replicated plasmid to just one => they behave as mini-chromosome. 47  3/30/2023 C) S. cerevisiae limitations for foreign protein expression: There is no strong inducible promoter and generally the product yields are low (max 1-5% of total protein) The presence of foreign gene products during the growth phase hinders growth. Plasmid instability is especially high when the foreign protein product is somehow toxic to the yeast. Also, secreted glycoproteins are hyperglycosylated => diminished activity. Many of the secreted proteins of S. cerevisiae are not found free in the medium, but rather in the periplasm. => purification problems and a decreased product yield. The gal promoter of yeast It is very strong and allows 1000 fold induction of GAL1 gene expression (0.8% of total yeast proteins). So, one can use galactose to induce the synthesis and glucose to repress it. 48  3/30/2023 (+) Galactose alone, which inhibits Gal80, Gal4 binds UAS and activates transcription. (+) Glucose, no Gal4 protein. GAL4 is complexed with GAL80 in the cytosol No Enter in the nucleus to shut off Gal1 expression. Cre-LoxP system in KO mice 49  3/30/2023 IV. Directed or targeted mutagenesis and protein engineering Random mutagenesis: Mutagens include – Ionizing radiations (e.g. X-rays, gamma rays), – Non-ionizing radiations (e.g. ultraviolet light) and – Various chemicals (e.g. mustard 65 gas, benzene, ethidium bromide), Used to produce random mutations in the DNA of organisms in attempts to improve them - a process known as strain improvement. IV. Directed or targeted mutagenesis and protein engineering Directed (Targeted) mutagenesis: Due to the use of recombinant DNA technology, mutagenesis can now be directed or targeted to a single base in the DNA sequence of a gene. => Effect on specific a.a in the protein These proteins are either created: Individually by site-directed mutagenesis Random mutagenesis : As large pool or library of millions of variants. The library is then screened or subjected to a special selection procedure to obtain the proteins with the desired characteristics. 50  3/30/2023 IV. Directed or targeted mutagenesis and protein engineering IV.1.A. Site-directed/ targeted mutagenesis (SDM): A molecular biology technique in which a point mutation is created at a defined site in a DNA molecule. In general, SDM requires that the wild type gene sequence be known. Three methods for carrying out SDM: Oligonucleotide-directed mutagenesis with plasmid DNA Phage M13-based methods PCR Phage M13-based methods 51  3/30/2023 Oligonucleotide-directed mutagenesis with plasmid DNA V.1. Problems associated with large scale bacterial cultures: V.1. Problems associated with large scale bacterial cultures: -Bacterial batch culture: It is the simplest way to produce recombinant proteins in bacterial cell cultures. All the nutrients required for cell growth are supplied from the start, and the growth is initially unrestricted. However, problems concerning growth to high cell densities are: oxygen limitation, reduced mixing efficiency, heat generation and high partial pressure of CO2. Also, certain metabolic pathways in the cell will be saturated, which potentially leads to the accumulation of inhibitory by-products (such as acetate) in the medium. 52  3/30/2023 V.1. Problems associated with large scale bacterial cultures: Therefore, only moderate cell densities and production levels can normally be obtained with batch cultures. Maintaining acetate concentration below the inhibitory level can be achieved by: – Limiting nutrients such as sources of carbon or nitrogen, – using glycerol or fructose instead of glucose as the carbon energy source, – adding glycine and methionine, – lowering the cultivation temperature etc. V.1. Problems associated with large scale bacterial cultures: -Bacterial fed-batch culture: It is commonly used in order to obtain high cell density and high protein production levels in bacteria. The carbon energy source is added in proportion to the consumption rate, so that, overflow metabolism and the accumulation of inhibitory by-products can be minimized. The bioreactor is preferentially equipped to maintain an optimal oxygen concentration, pH and temperature. 53  3/30/2023 V.2. Problems associated with large scale mammalian cultures: The culture of animal cells is much more delicate than that of micro-organisms: they are more fragile, reproduce more slowly and require, in many cases, support to develop Key barriers to large-scale mammalian cultures include: oxygen supply limitations, waste product accumulation, shearing sensitivity of animal cells, and The challenges of growing adherent cell lines. Chapter 4: Uses of Microbial Systems in Molecular Biotechnology Pr. Said El Shamieh B3212 Molecular Biotechnology Bachelor of Biology 54  3/30/2023 I. Molecular diagnostics The success of modern medicine depends on the detection of small molecules in water, plants, soil and human. A good detection system should have three qualities: Sensitivity which is the ability to detect very small amounts of target even in the presence of other molecules, Specificity which means that the test yields a positive result for the target molecule only, and Simplicity which is to run the test efficiently, inexpensively and on a routine basis. I. DNA diagnostic systems: Diagnosis of genetic diseases: b- Polymerase Chain Reaction (PCR): PCR can be used as a prognostic tool to determine viral load and to determine the effectiveness of antiviral therapy. Specific primers are now available for the detection of many pathogens including bacteria (E. coli and M. tuberculsosis), viruses (HIV) and fungi. 55  3/30/2023 II. Therapeutic agents and vaccines Before the advent of molecular biotechnology most human proteins were available in only small (limited) quantities. Today, hundreds of genes for human proteins have been cloned, expressed in host cells and tested as therapeutic agents (drugs) in humans. II.1. Pharmaceutical products and enzymes II.1.a. Insulin: In mammals, insulin is expressed as a single chain pre-pro-hormone, which is secreted through the plasma membrane. This prepro- hormone contains extra amino acids not present in the mature hormone: Pre-pro-insuline Pro-sequences present before the pre-sequence and target the expressed protein for secretion the pre-sequence present in the middle (peptide C) of the chain is important for the function. During secretion through the plasma membrane, these extra amino acids are cleaved from the prehormone by cellular proteases to release the mature insulin molecule consisting of two short polypeptide chains, A and B, linked by two disulphide bonds. Pre-prohormone 56  3/30/2023 The primary problem in the production of insulin in bacteria was getting insulin assembled into this mature form. Creation of a single glycosidase-insulin fusion protein which could be cleaved in a single step to release the mature insulin from the b-galactosidase peptide: The A and B chains of insulin are first expressed separately in E. coli as fusion proteins with B- gal, Then the fusion proteins are cleaved from B-gal The purified A and B chains are mixed and reconnected in a reaction that forms disulfide cross bridges. II.1.b. Enzymes as therapeutic agents: Cystic fibrosis (CF) is a fatal hereditary disease caused mutations in the CFTR gene. A thick mucus (making breathing difficult) forms in the CF individuals as a result of alginate which is a polysaccharide polymer produced by the bacterium P. aeruginosa, DNA from lysed cells and leukocytes which accumulate due to the infection. Scientists isolated the gene for DNase1, purified the enzyme and delivered it as an aerosol to the lung where it hydrolysed the DNA into short oligonucleotides and decreased viscosity in the lungs making breathing easier. Combined with DNase1, alginate lyase which is an enzyme that can liquefy bacterial alginate, is used. 57  3/30/2023 II.2. Attenuated vaccines II.2. Attenuated vaccines (or live vaccine): Attenuated vaccines often consist of a pathogenic strain in which the virulent genes are deleted or modified. Example: cholera. The problem with attenuated vaccines is that sometimes these viruses are not killed or are not weakened enough, thus they end up causing the disease and sometimes even death. V. cholerae produce an enterotoxin with an A subunit and 5 B subunits. A live vaccine was developed by mixing a plasmid containing a modified A peptide (inactive) with a V. cholerae that contains a tetracycline resistance gene inside the A gene sequence in its chromosome. By recombination, the modified inactive A peptide replaces the tetracycline insert in the Vibrio chromosome. 58  3/30/2023 II.3. Subunit vaccines: viral antigenic or microbial proteins instead of the whole organism are used. The first successful subunit vaccine was produced for hepatitis B virus (HBV), which infects the liver. II.4. Vector vaccine: It is a vaccine which is introduced by a vector e.g. vaccinia virus. The virus replicates is generally nonpathogenic and its genome has been completely sequenced. These characteristics make the vaccinia virus a good candidate for a virus vector to carry genes for antigenic determinants form other pathogens. The vaccinia virus as a live vaccine led to the global eradication of the smallpox virus. 59  3/30/2023 III. Synthesis of commercial products by recombinant microorganisms III.A. Synthesis of restriction endonucleases and small biological molecules 1- Cloning and production of restriction enzymes: Cloning of the RE Pst1 of Providencia stuartii into E.coli The chromosomal DNA of Providencia stuartii is digested with HindIII Ligated into HindIII site of the E. coli plasmid vector pBR322 The genomic library is transformed into E. coli and grown in liquid culture The transformed E. coli cells are infected with lambda phage. Only lysis resistant colonies will grow: If Pst1 is expressed in E. coli these cells will become resistant to the lytic action of lambda, because the enzyme will degrade the DNA of the infecting lambda phage. Finally, the lambda-resistant transformants are grown and assayed for Pst1 activity 60  3/30/2023 2- Synthesis of Vitamin C (L-Ascorbic acid): It is synthesized by a very expensive process which includes a microbial fermentation step and a number of chemical steps which convert glucose to ascorbic acid, with the last step involving conversion of 2-keto-L- gluconic acid (2-KLG) to L-ascorbic acid. Strategy: The gene for the enzyme 2,5-DKG reductase was purified from Corynebacterium, cloned into E. coli first and then subcloned and expressed in Erwinia herbicola. The recombinant Erwinia cells were able to convert glucose to 2-KLG. Erwinia herbicola Corynebacterium Chapter 5: Plants Biotechnology Pr. Said El Shamieh B3212 Molecular Biotechnology Bachelor of Biology 61  3/30/2023 I.1. Tissue cultures and transformation techniques of plants I. GENETIC MANIPULATION AND ENGINEERING OF PLANTS a. Culturing and sub-culturing of plant cells: Plant cell cultures are typically grown as cell suspension cultures in liquid medium or as callus (undifferentiated lump of cells) cultures on solid medium. The culturing of undifferentiated plant cells requires the proper balance of the plant growth hormones, auxin and cytokinin. – An excess of auxin will often result in a proliferation of roots – An excess of cytokinin may yield shoots, and 62  3/30/2023 b. The plant model systems for gene- transfer experiments: Arabidopsis thaliana cells: They are isolated from Arabidopsis which is the tiny, rapidly growing member of the mustard family. This plant is well-suited to genetic analysis of a variety of developmental and physiological processes. It takes up little space, easy to grow, has a small genome, and its genes defined by mutations. Introducing genes is done using the bacterium Agrobacterium tumefaciens which readily infects dicots but not monocots. 63  3/30/2023 I.1.A. Culture types and regeneration of plants The tissue obtained from the plant to be cultured is called an explant. The most commonly used tissue explants are: The meristematic ends of the plants like the stem tip, The auxiliary bud tip The root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins. a. Culture types: 1. Protoplasts: Protoplasts are plant cells with the cell wall removed. They are most commonly isolated from either leaf mesophyll cells or cell suspensions. Removing the cell wall can be done mechanically or enzymatically. Enzymatic isolation is usually preferred and carried out in a simple salt solution containing cellulase and pectinase enzymes. 64  3/30/2023 Culture types: 2. Root cultures: Root cultures can be established in vitro from explants of the root tip using fairly simple media. 3. Shoot tip culture: The tips of shoots can be cultured in vitro, producing clumps of shoots. 4. Embryo culture: Embryos (zygotic) can be used as explants to generate callus cultures or somatic embryos. Culture types: 5. Microspore culture: Haploid tissue can be cultured in vitro by using pollen or anthers as an explant. Anthère: Partie du stamen ou’ le pollen est produit. 65  3/30/2023 I.1.B. Agrobacterium-mediated gene transfer: The plasmid Ti and its derived vectors a. Methods of infecting plant cells with Agrobacterium: Three general methods are available for obtaining Agrobacterium transformed plant tissue: 1) Plants are grown under aseptic conditions and the stem is wounded. The wounded tissue is inoculated with the cells of A. tumefaciens by means of a syringe. The tumors that develop can be removed and cultured on hormone free agar on which the transformed cells can grow. 2) Co-culturing. Protoplasts lacking cell walls are first produced by dissolving the cell wall enzymatically. The protoplasts are then allowed to remain for about two days so that cell walls begin to form. At this stage (usually between 36-48 h) A. tumefaciens suspension is added. Some of the cells undergo transformation during the next few days of co-culturing. Then by adding antibiotics, the bacterial cells are killed and the plant cells can be grown as callus on hormone free agar medium. 3) Leaf disk method in which large pieces are cut and incubated with Agrobacterium so that wounded cells at the cut edges become genetically transformed. Agrobacterium-mediated plant transformation Agrobacterium genus : – soil bacteria that infects wounded plants & leads to gall formation – induces tumors by genetically Eng’g plant cells at the wounded site – T-DNA acts as an insertional mutagen – GMO Agrobacterium tumefaciens & crown gall tumor 66  3/30/2023 b. The Ti plasmid of Agrobacterium: Tumor-inducing plasmid (Ti) : a 235 kbp vector which harbors virulence (vir) genes, organized in 10-12 operons and encoding bacterial factors for the DNA transfer, and oncogenic T-DNA required for tumorogenesis. Virulence genes LB RB T-DNA introduced into plant Xmal DNA ~200 Kb LB Selectable marker RB Ti plasmid T-DNA plasmid for transformation c. Using the Ti plasmid of Agrobacterium as a vector for cloning of foreign DNA: Due to its large size, direct cloning into the Ti plasmid is impossible. More important, vir and T-DNA, do not have to be on the same plasmid. Hence it is useful to perform all manipulations on an excised piece of DNA including the T-DNA and then use in vivo recombination to swap the engineered T-DNA for its normal version in an intact Ti plasmid. 67  3/30/2023 The cloning strategy 1- Cloning the T-region of Ti plasmid into an E. coli plasmid: The T-region is cut out of the Ti plasmid with restriction enzymes and introduced into one of the standard cloning vector plasmids that are used with E. coli. 2- Using restriction enzymes and recombinant DNA techniques to insert a foreign gene into the T-DNA of the hybrid E. coli plasmid, and making large amounts of this hybrid plasmid in E. coli. 3- Introducing this hybrid plasmid into A. tumefaciens cells containing the corresponding entire Ti plasmid. Homologous genetic recombination between the cloned T-DNA segment carrying the foreign gene and the T-DNA segment of the native Ti plasmid results in transfer of engineered T-DNA to the Ti plasmid and displacement of the normal T-DNA.  The outcome is an engineered A. tumefaciens with a Ti plasmid whose T-region now carries the goi 4- Infect plants with these engineered A. tumefaciens bacteria. The crown gall cells that result will be transformed by the T-DNA carrying the foreign gene. Ti plasmid the role of Agrobacteria is solely that of a vector to bring T-DNA into plant cells. 68  3/30/2023 e. 3. Basic characteristics and vector optimization for plant transformations Different types of binary vectors have been devised to suit different needs in a plant transformation process. Reporter gene: Some vectors are equipped with reporter genes as a screening tool. The most utilized reporter gene so far is uidA, which formes an easily identifiable blue precipitate. Alternatively, GFP. Plasmid size: Most of the plasmids employed in plant transformation range from 15 to 25 kb but the construction of an artificial binary bacterial chromosome (BIBAC) made the stable insertion of 150 kb possible. Promoters: Constitutive promoters, like 35S from cauliflower mosaic virus (CaMV), have been widely used as single or double copies. II. METHODS OF DIRECT TRANSFER OF GENES: 69  3/30/2023 A- Biolistics (biological ballistics): Also called microprojectile acceleration or microparticle bombardment. This transformation method consists of the acceleration of a macroprojectile loaded with millions of microparticles which are tungsten or gold microspheres about 1 μm in diameter. The microspheres are coated with the goi, and have a high specific mass, allowing them to acquire the needed momentum to penetrate the target cells. Helium gas at high pressure is used to propel the microparticle. Once inside the target cells, the DNA coating the microspheres is released and can be integrated into the plant's genome. This technique is generally less efficient than Agrobacterium-mediated transformation. b- Microinjection: This method was developed for animal transformation but has also been extended to plants. In this technique, microcapillary needles are used to introduce DNA directly into cells. Each cell to be transformed must be manipulated individually. 70  3/30/2023 c- Electroporation: It is a process whereby very short pulses of electricity are used to reversibly permeabilize the lipid bilayers of plant cell membranes. The electrical discharge enables the diffusion of macromolecules such as DNA. Because the plant cell wall will not allow efficient diffusion of many transgene constructs, protoplasts (without cell walls) must be prepared. d- Direct transformation with the use of polyethylene glycol (PEG): This is a simple method that consists of adding great amounts of transgenic plasmids to a protoplast culture, which guarantees that a small proportion of the protoplasts will take up and assimilate the plasmids. The assimilation rate can be increased with the addition of PEG. III. PRODUCTION OF “CLEAN”, MARKER-FREE TRANSGENIC PLANTS 71  3/30/2023 The presence of selectable marker genes which are essential for the initial selection of transgenic plants is seen by regulatory agencies in Europe as undesirable. One of the main causes of concern is the possible spread of antibiotic and herbicide resistance genes into the environment. => A need for the development of techniques for the efficient production of "clean", marker-free transgenic plants. 1- Molecular cut and paste: site specific recombination: The transformed plant cells are generated in such a way to contain: the gene encoding the microbial recombinase and the marker gene, both of which flanked by Cre / Loxp. Bacteriophage P1 DNA Palindromic sequence LoxP 72  3/30/2023 2- The use of alternatives markers: Transgenic events can be selected using markers that enable them to use a particular food source, instead of using antibiotic/herbicide resistance makers. Other selection systems include: – Genes that allow the growth of plant cells in the presence of a particular sugar or sugar alcohol as their sole energy source (e.g. xylose, arabitol, glucose). – Genes that allow plants to survive in media supplied with amino acid analogs. The use of alternative markers completely eliminates concerns over the possible spread of antibiotic and herbicide resistance genes into the environment. However, since these markers entail the introduction of new metabolites, a more rigorous risk assessment will be needed to establish the safety of the resulting transgenic products. IV. THE PLANTS AS BIOREACTORS 73  3/30/2023 IV. The plants as bioreactors The first plant-produced pharmaceutical product, the human growth hormone, was produced in transgenic tobacco in 1986. For producing recombinant proteins, plant bioreactors: Are cost-effective and easy for agricultural scale-up have post-translational modifications lack contamination by animal pathogens. One of the major obstacles in the plant bioreactor is the low yield of recombinant proteins which are susceptible to proteolytic degradation in transgenic plant cells. Potential approaches to improve the yield of recombinant proteins include: Reducing protease activity in transgenic plants Using proteinase inhibitors (PIs) which can be added directly to transgenic plants, cultures and/or to protein extraction. A- The seed-based bioreactor platform: A.1. The protein storage vacuole (PSV) as bioreactor: Most soluble proteins in seeds are stored in a specific compartment termed PSV or protein body to be used upon seed germination. PSV in most seeds contains three morphologically and biochemically distinct subcompartments: the matrix, globoid and crystalloid. The matrix is the major deposit tank of soluble storage proteins. Specific targeting signals, such as the AFVY vacuolar sorting determinant of phaseolin, can deliver reporter or recombinant protein to PSV matrix that is separated from the globoid and crystalloid. 74  3/30/2023 A.2. Oil body (OB) as bioreactor: Fusion proteins containing oleosin are successfully targeted and stably accumulated on seed OBs. The use of OBs as storage organelles in plant bioreactors has many advantages. Firstly, OBs exist in diverse tissues including seeds, pollen and fruits but especially enrich in oilseeds. OBs can be separated from other cellular components by using a flotation centrifugation method. V. APPLICATIONS OF GM PLANTS Several concerns are associated with the use of herbicide- resistant crops. These include: – drift to nearby susceptible vegetation, – herbicide-tolerant crops becoming weedy and difficult to control (because glyphosate-resistant weeds have developed), and increased selection for resistant weed species or shifts in weed populations. – Link of iron deficiencies in some crops to widespread glyphosate usage in some studies: having potential economic and health implications. 75  3/30/2023 E- Improvement of crop quality: Figure 12.33b: Rice plants with both parts of the pathway produce grains with a yellowish cast (top) due to the β-carotene they contain, in contrast to the pure white grains (bottom) of normal plants. 151 Courtesy of Ingo Potrykus, Institute für Pflanzenwissenschaften, ETH Zurich. Chapter 6: Transgenic animals Pr. Said El Shamieh B3212 Molecular Biotechnology Bachelor of Biology 76  3/30/2023 I. Methods for Producing GMOs I.1. DNA microinjection: This method involves: – Direct microinjection of a chosen gene construct into the pro-nucleus of a fertilized ovum. The insertion of DNA in the host genome is, however, a random process. – The manipulated fertilized ovum is transferred into the oviduct of a female recipient or foster mother. A major advantage of this method is its applicability to a wide variety of species. I.2. Gametes-mediated DNA transfer: Sperm-mediated transgenesis (SMGT): Sperm cells are able of carrying exogenous DNA into the oocyte during fertilization. In the original protocol, spermatozoa are incubated with the DNA containing the gene of interest followed by in vivo or in vitro insemination. DNA binds to the sperm’s plasma and part of it (15-20%) is internalized by a mechanism mediated by CD4 molecules and carried into the oocyte upon fertilization. 77  3/30/2023 I.1. DNA microinjection: I.2. Gametes-mediated DNA transfer: Sperm- mediated transgenesis (SMGT): The most appealing characteristics are: Its simplicity, as embryo manipulation is not required, The possibility of performing mass production of genetically modified animals through in vivo or in vitro insemination of many oocytes. High efficiency (up to greater than 80%) and relatively inexpensive Can be used in species refractory to microinjection whenever reproduction is mediated by gametes. 78  3/30/2023 3. Embryonic stem cell-mediated gene transfer: This method involves: prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Transgenic ES cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell. At this stage, embryos carrying the transgene can be frozen and stored for subsequent implantation. One big advantage of using ES cells over microinjection is the ability to select for transgene integration through the use of selectable markers. The use of ES cells also allows for the targeted alteration of DNA by homologous recombination, making it the method of choice for gene inactivation. 79  3/30/2023 Transplantation or nuclear transfer: Somatic Cell Nuclear Transfer (SCNT) : It is referred to as nuclear cloning, nuclear transfer or nuclear transplantation. It involves: Isolating a nucleus from an adult donor cell (somatic) introducing the somatic nucleus into an enucleated oocyte Stimulating cell division by heat shock Transferring it to the uterus of a female recipient animal In the egg, the somatic cell nucleus is reprogrammed by the host cell. When transferred to the uterus of a female recipient, the resulting cloned embryo has the potential to grow into an infant that is a clone of the adult donor cell, a process termed “reproductive cloning.” In culture, the embryo derived from nuclear transfer can also give rise to embryonic stem cells which can potentially be used in regenerative medicine, sometimes referred to as "therapeutic cloning." The success rate for SCNT averages 1-3% in most animals, which is considered low. Transplantation or nuclear transfer: Somatic Cell Nuclear Transfer (SCNT) : Animal clones are not “biotech” or “genetically engineered” animals but instead they are “conventional” animals. SCNT has facilitated the ability to make transgenic and knockout animals by first introducing transgenes. The proper use of SCNT ensures that every cell of a cloned animal will have the transgene. SCNT is allows homologous and non homologous recombination. 80  3/30/2023 II. Transgenic mice II.A. Genetic modifications by the system of recombination Cre-loxP: Used for precise removal of DNA, such as eliminating an endogenous gene (conditional knock out) or transgene or activate a transgene. The Cre/loxP system is a very useful tool to study their function in differentiated tissues, especially when dealing with genes required early in embryonic development and later in differentiated tissues. 81  3/30/2023 II.B. Conditional control of gene expression in transgenic mice: The ability to turn genes on and off at will has been achieved by using binary systems in which gene expression is dependent on the interaction of two components, resulting in either transcriptional transactivation or DNA recombination. II.B.3. Transgenic mice as models for human diseases: Human genetic diseases are caused by numerous types of mutations. Models for human disease have been made by mutating the same gene in mice that is responsible for the human condition. In most cases, these models replicate many of the corresponding human disease phenotypes. Animal models have greatly improved our understanding of the cause and progression of human genetic diseases and have proven to be a useful tool for discovering targets for therapeutic drugs. 82  3/30/2023 Chapter 7. Stem Cells Pr. Said El Shamieh B3212 Molecular Biotechnology Bachelor of Biology Overview of the birth, lineage, and death of cells 166 83  3/30/2023 I. Sources and properties of different types of stem cells Classically, a stem cell is defined as cell that possesses two properties: self-renewal: which is the ability to renew themselves through mitotic cell division, and potency: which is the ability to differentiate into a diverse range of specialized cell types. I.1. Classification of stem cells according to their genetic potential: Potency of a stem cell is its capacity to differentiate into specialized cell types: a- Totipotent stem cells are cells that can differentiate into embryonic and extra-embryonic cell types. Such cells are produced from the fusion of an egg and sperm cell and can construct a complete viable organism. The cells that are produced by the first few divisions of the fertilized egg (morula stage) are also totipotent. 84  3/30/2023 b- Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers. Cells of the inner cell mass of the blastocyst stage. These cells can give rise to the three embryonic germ layers. c- Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells. Embryonic stem cells can be maintained in culture and form differentiated cell types 170 85  3/30/2023 Fates of the germ layers in animals 171 d- Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells. e- Unipotent cells can produce only one cell type (e.g. muscle stem cells or neuronal stem cells), their own, but have the property of self-renewal which distinguishes them from non- stem cells. 86  3/30/2023 I.2. CLASSIFICATION OF STEM CELLS ACCORDING TO THEIR STATE OF DEVELOPMENT: 87  3/30/2023 a- Embryonic stem cells: They are derived from the epiblast tissue of the ICM Pluripotent and give rise during development to all derivatives of the three primary germ layers. Nearly all research to date has taken place using ESCs. mESCs are grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF). hESCs are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic Fibroblast Growth Factor A- Embryonic stem cells Without optimal culture conditions or genetic manipulation, ES cells will rapidly differentiate. A hESC is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency. ES cells, being pluripotent cells, require specific signals for correct differentiation. Es cells are a potential source for regenerative medicine and tissue replacement after injury or disease. 88  3/30/2023 B- Adult stem cell: An adult stem cell refers to any cell which is found in a developed organism. They can be found in children as well as adults. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, and maintain the normal turnover of regenerative organs such as blood, skin, or intestinal tissues. Pluripotent adult stem cells are rare and generally small in number. The use of adult stem cells in research and therapy is not as controversial as embryonic stem cells. Additionally, because some instances adult stem cells can be obtained from the intended recipient, (an autograft) the risk of rejection is essentially non-existent in these situations. Regeneration of the intestinal epithelium from stem cells 178 89  3/30/2023 II. RETRO-DIFFERENTIATION AND TRANS-DIFFERENTIATION II.A. Retro-differentiation When mature, specialized cells can revert back to a more primitive, immature cell stage. In an approach called induced pluripotency. These are reprogrammed cells (e.g. epithelial cells) given pluripotent capabilities. Genetic reprogramming of cells has been carried out using the transcription factors Oct3/4, Sox2, c- Myc, and Klf4. A major advantage is that it can generate cells of the specific arm of the immune system, such as B and T lymphocytes that can help to fight viral infections and cancer. 90  3/30/2023 I.B. Trans-differentiaion It takes place when a non-stem cell transforms into a different type of cell, or when an already differentiated stem cell creates cells outside its already established differentiation path. It is an irreversible switches of one differentiated cell type to another. E.g: in salamanders and chickens, cells of the iris turn into lens cells when the lens of the eye is removed. III. TREATMENT OF HUMAN DISEASES BY CELL THERAPIES 91  3/30/2023 Current treatments: For over 30 years, bone marrow and more recently umbilical cord blood stem cells have been used to treat cancer patients with conditions such as leukemia and lymphoma. During chemotherapy, most growing cells are killed by the cytotoxic agents. It is this side effect of conventional chemotherapy strategies that the stem cell transplant attempts to reverse; a donor's healthy bone marrow reintroduces functional stem cells to replace the cells lost in the host's body during treatment. V. Scientific, medical, social and ethical aspects the possible risk that transplanted stem cells could form tumors and have the possibility of becoming cancerous if cell division continues uncontrollably. But the major stem cell research controversy arises from the ethical debate centered specifically on research involving the creation, usage and destruction of human embryos (or abortion). 92