Applications of genetics v2

Applications of genetics v2

1. Genetic engineering

   • Altering/moving genes to make GM organism
     • Transfer genes into:
       • Bacteria, so they make useful products (Insulin)
       • Plants/animals: Acquire characteristics
         • Humans: Reduce effects of genetic disease
   • When genetic material from 2 species is combined
     • Results in recombinant DNA = DNA from 2 species
       • Transgenic: Organism that has been genetically modified
         • By the addition of a gene/genes from another species
   • Introduced DNA = Donor DNA
     • Organism DNA introduced to = Host
       • When the host incorporates foreign gene
         • It is said to have transformed
   • Organisms that have genes altered/removed aren't transgenic as no foreign genes present
   • DNA identification & isolation
     • 1) Locating the gene
       • Only 1/20,500 code for insulin
         • Gene identified w/gene probe
           • Specific segment of single stranded DNA that's complementary to a section of the gene
     • 2) Isolating the gene
       • Using Restriction Endonuclease
         • Are bacterial enzymes that cut DNA at specific nucleotide sequences
           • As sequences occur in many places, DNA is cut into smaller fragments
             • & individual genes can be isolated
               • Some Restriction enzymes cut straight across, making a blunt cut
                 • Restriction Enzymes
                   • Bacterial enzyme that cuts the sugar-phosphate backbone at a specific nucleotide sequence
                 • Many make a staggered cut which leaves unpaired bases on both strands = Sticky Ends
                   • E.g EcoR1 in E.coli
         • Drawbacks
           • If the recognition sequence occurs within the gene of interest, fragments will have no function
           • Eukaryotic genes contain introns, which are normally removed when RNA is made
             • But inserting an entire gene means that introns won't be removed
               • & bacteria don't have introns therefore can't process them
                 • Meaning translated proteins will have non-functional amino acids
       • Using Reverse Transcriptase
         • Enzyme derived from a retrovirus, that catalyses the synthesis of Copy DNA (cDNA) from and RNA template
           • DNA polymerase catalyses synthesis of DNA complementary to cDNA
             • Making double-stranded molecule containing gene for insulin
         • Doesn't have the problem of introns
     • 3) Inserting recombinant plasmid into vector
       • Bacterial cell is unlikely to take a gene in spontaneously
         • So gene is carried into cell via a vector
           • Viruses = Classic example, but in example plasmid = vector
             • Effective as plasmid can move in/out of cells
       • To isolate plasmids, bacteria is treated with:
         • EDTA to destabilise cell walls
         • Detergent to dissolve cell membrane
         • NaOH to make alkaline environment, denaturing membrane proteins
         • Plasmids removed from cell debris, & cut open w/same restriction endonuclease
           • Meaning it has same nucleotide sequence in its sticky ends
             • Vector & gene mixed, complementary base sequences pair
               • Gene becomes loosely bound to the plasmid
                 • DNA Ligase makes join permanent by joining both sugar-phosphate backbones together
               • WAGOLL vector
                 • Self-replicating
                 • Small
                 • Not broken down by host enzymes
                 • Not stimulate immune response in recipient
                 • Be able to be screened, to confirm gene was actually inserted
                 • Have markers, so that host cells that have taken up the gene can be identified
     • 4) Transfer DNA into host cell
       • When bacteria & plasmids mixed only 1% combine
         • Increased by adding CaCl2, the +ve Ca binds the -ve DNA backbone of plasmid w/membrane lipopolysaccharides
           • Plasmid passes into cells w/heat shock (Cells chilled to 4'c are briefly heated to 42'c)
     • 5) Identify host cell containing recombinant w/gene markers
       • To check if gene is successfully incorperated
         • Vector can have DNA sequenced
         • Plasmids w/antibiotic resistant genes are used
           • & grown in a culture with the anitbiotic
             • If they continue to grow then resistance gene is present
               • The resistance gene is a marker gene because it marks the presence of the plasmid
         • To distinguish which transformed bacterial cells have taken up empty plasmids
           • Blue-white screening is used, bacteria grown on medium w/lactose analogue X-gal
             • Turn white if present, Blue if not present
       • Bacteria w/recombinant plasmids are cultured in large volumes in fermenters making a clone
         • Clone= Population of genetically identical cells/organisms formed from a single cell/parent respectively
         • When DNA transcribed insulin is made & collected
     • Description of human insulin into E.coil
   • Pros
     • Medical products
     • Tooth Decay
     • Prevention & treatment of diesease
     • Enhancing crop growth
     • Environmental use
     • Proteins can be made in high qunatites
   • Cons
     • Plasmids are easily transferred
     • May activate oncogenes
     • Might get into the environment & become a threat
     • Affect the functions of other genes
   • Genetically Modified Crops
     • Introducing gene into plant cell
       • Gene gun fires small spheres (Gold/Tungsten)
         • Coated w/preparation of gene at plant cells
           • Some penetrate cell wall & pass through cell membrane
       • Electroporation: Electric field increases permeability of cell membranes enhancing gene uptake
       • Microinjection: Membrane is pierced w/a fine needle, injecting gene into cytoplasm/nucleus
       • Most common method: Use bacterial vector Agrobacterium tumefaciens
     • Transforming plants w/Agrobacterium tumefaciens
       • Infects plant & inserts t-DNA into plants genes
         • T-DNA = section of plasmid
         • See diagram pg 214
         • T-DNA normally stimulates tumour growth
           • But this region is replaced w/useful genes
       • Soya beans
         • Used to make flour, protein, oils
           • They have herbicide resistant gene
             • So when weed-killer is sprayed soya growth isn't inhibited
       • Bt Tomatoes
         • Bacillus thuringiensis has a gene that codes for insecticides
           • Gene has been inserted into tomato leaf cells, humans not affected
       • Antisense Tomatoes
         • Grown 1994-97, not a commercial success
           • Another copy of the polygalacturonase gene added
             • This made a double-stranded molecule that stopped the breakdown of pectin
     • Arguments for/against GM crops
       • For
         • Higher Crop yield: Adding genes for insect/fungus/drought/salt/herbicide resistance helps increase yield
         • Pesticide reduction: genes for pathogen resistance also reduce quantity of pesticides needed
         • Improved food: Nutritional quality can be enhanced (Golden rice, beta carotene)
         • Pharming: Making pharmaceutical molecules in genetically modified crop plants
       • Against
         • Pollen from GM plants may transfer to wild relatives, could make 'superweeds'
         • Pest resistance: Plants w/insect resistant genes, Insects might become resistant to it
         • Marker genes: Gm organisms contain marker genes that might transfer to bacteria in the consumer
         • Plant breeding might become a monopoly, reducing diversity of crops
         • New protein: Expression of genes making new protein may have adverse affect on health
         • Organic farming: Pollen from GM crops could compromise organic crops
         • Economic expense and obeying laws

2. Genetic screening & Gene therapy

   • Caused by abnormalities in genome
   • Genetic Screening
     • Used to
       • Confirm diagnosis
       • Indicate appropriate treatment
       • Prevent families having kids w/devastating conditions
       • Identify those w/high risk of condition (Carrier screening)
       • Cons
         • Could invade privacy
         • Can lead to more abortions (Tend to be female)
         • Could be put in high cost insurance groups
       • Pre-natal diagnostic testing/newborn screening
       • Pre-symptomatic testing, for adult onset diseases/disorders
       • Forensic/Identity testing
     • Commercial gene tests
       • Give probability of developing some conditions
       • Limited to
         • Companies aren't regulated/verified
         • Test on a small proportion of geneome
         • Difficult to interpret result: Might have mutation for disease but never develop it
         • Lab errors
         • No medical options available for some diseases
         • Might provoke anxiety/social discrimination/stigmatisation
     • Cystic Fibrosis (CF)
       • Homozygous for autosomal recessive allele, identifiable via blood test
         • Normally cystic fibrosis transmembrane regulator (CFTR)
           • Transports Cl- & Na+ out of cell, H20 follows by osmosis, keeping mucus waterey
             • Mutant CFTR can't transport ions so mucus remains thick & sticky
               • Bronchioles & alveoli clog up, caused difficulty breathing/ recurrent infections
               • Pancreatic duct becomes blocked, food not fully digested (No pancreatic enzymes)
               • Vas deferens become blocked
               • Liposomes are inhaled, fusing w/phospholipid bilayer of lung epithelial cells
                 • DNA enters cells, transcribed making CFTR, provides relief, not passed as genes are replaced
                   • This is treatment not a cure
   • Gene therapy
     • To introduce vector, gene therapy uses
       • Virus/Plasmid as vector
       • Injection of naked plasmid DNA
     • 2 Main approaches
       • Somatic cell therapy
         • Treating body cells in affected tissues
           • Doesn't appear in offspring
             • Process: Lysosomes > Endocytosis > Myci cells of lipids
       • Germline therapy
         • Replacing genes in oocyte
           • Gene correction inherited > Made permanent & passed to offspring
             • Controversial, can affect other genes, unpredictable effects
   • Duchenne Muscular Dystrophy (DMD)
     • Recessive, sex-linked
     • Caused by 1 or more deletions in dystrophin gene
       • Affects reading frame, dystrophin is a structural protein,
         • So suffer muscle loss, become wheelchair-bound by teens
           • Drisapersen is an antisense oligonucleotide drug
             • Has 50 nucleotide sequence that's complementary to mutated sequence
               • Treats DMD by acting as molecular patch, binds w/mRNA over the exon w/the deletion
                 • This portion of RNA therefore becomes double-stranded, so ribosome can't translate it
                   • Restoring reading frame so partially functional dystrophin is made
                     • Called exon skipping, delivered by subcutaneous injections
                       • Gene therapy via virus/ stem cell research are alternatives
   • Effectiveness
     • Advantages outweigh disadvantages
       • But only a small portion of introduced genes are expressed
       • May be immune reponse in patient
   • Genomics & healthcare
     • Genomics: Concerning the structural & functional analysis of genomes
       • Improvements in healthcare
         • More accurate diagnosis
         • Better prediction of the effect of drugs
         • New/improved treatments
         • NGS can help give custom dosages (E.g Warfarin)

3. Tissue Engineering

   • Used to repair/improve/replace biological functions, makes bio-artificial organs/regenerate tissue
   • Cells for tissue engineering
     • Telomeres are the repeated nucleotide sequences on the end of chromosomes
       • Which short at each division limiting cell mortality
         • In 1998 process to elongate them was developed, extending life of differentiated cells
           • Cells separated w/enzymes (trypsin), elongated & used to grow tissue replacements
             • E.g Fibroblasts = Skin Replacement, Chondrocytes = Replacing Cartilage
               • Stem cells are preferred as they can be extracted from blood/solid tissue
   • Cells are classified by source
     • Autologous cells: From same individual
       • Fewest problems w/rejection & pathogen transmission
         • But not always available (Too ill)
     • Allogeneic cells: From donor of same species
     • Xenogenic cells: From different species
     • Syngenic/Isogenic cells: From genetically identical cells
     • Tissue culture
       • Cells grown in tissue culture form cell lines that are clones
         • All genetically identical
           • Cell lines used to make cloned tissue samples/organs
             • Therapeutic cloning = Generating cloned material
               • Reproductive cloning: Cloning whole organism
                 • Law permits therapeutic but not reproductive
       • During period of growth
         • Cell needs to be given
           • Oxygen, Nutrients, Growth factors, Right: pH, Humidity, Temp, W.P
             • Diffusion happens in small structures, larger structures need capillary networks
       • Sometimes special physical/chemical stimulus is needed
         • E.G
           • Chondrocytes > low oxygen conc > skeletal tissue
             • Endothelial cells > shear stress > mimic blood flow in vessels
               • Heart values > Pressure pulses
   • Scaffolds
     • Cells are 'seeded' onto an artificial structure that supports 3d tissue
       • It must
         • Allow cells to attach & move
         • Deliver/Retain cells/molecules
         • Porous to allow diffusion of nutrients/waste
         • Be biodegradable & absorbed by surrounding tissues
           • Rate of degeneration must match rate of tissue formation, eventually forming neotissue

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