WJEC A2 Biology – Gene Therapy – Flashcards
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What is cystic fibrosis?
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A genetic disease affecting the exocrine glands. It causes the production of abnormally thick mucus, leading to the blockage of the pancreatic ducts, intestines, and bronchi and often resulting in respiratory infection.
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Why does CF cause a respiratory problems?
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Missing triplet in the gene coding for chloride pumps CFTR protein not formed properly Chloride ions are not pumped to the inside surface of the lungs/out of epithelial cells The water potential of the mucus is not lowered, so water does not enter the mucus from the cell by osmosis Causes the mucus to be thick and sticky Inhibits respiration (as moist surface req. on alveoli for gas ex.)
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What is the aim of gene therapy?
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To treat a genetic disease by replacing defective genes in a patient with copies of a new DNA sequence or by supplementing defective genes with non defective ones
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State two possible methods for replacing defective genes
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Somatic cell therapy and germ line therapy
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What are the day to day treatments of CF?
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Antibiotics used to fight off infections Inhaling enzymes that break down the mucus allowing it to be coughed up Steroids Diet high in calories and rich in fat and protein as not much absorbed Insulin and other drugs to treat symptoms of CF like diabetes Daily chest physiotherapy massage to keep airways open
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Define: Gene therapy
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The deliberate 'repair' or replacement of damaged genes
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Describe how germ line therapy works
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Replacement of genes in a blastocyst or eggs/sperm Blastocyst/gametes removed and screened Cell from blastocyst removed/gametes isolated Cell/gametes are treated by insertion of new gene IVF (rest of a blastocyst can be disposed of) and (re)implantation
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Describe the advantages and disadvantages of germ line therapy
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+ Child is born completely healthy with no chance of having the disease (no symptoms) - Ethical issues with altering blastocysts
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Describe somatic cell therapy
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Healthy allele is added to the affected cells (eg via an asthma pump). Supplementation This requires repeated application of the gene as the cells have limited life spans and do not pass the supplemented "healthy" allele onto the next generation
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State two delivery methods for somatic cell therapy used to treat CF in the lungs
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Using (adeno)viruses which have had the healthy CFTR allele inserted Using liposomes containing plasmids with the healthy CFTR allele Both delivery methods sprayed into lungs
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What are some symptoms of CF?
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Build up of mucus in the lungs, pancreatic duct and sperm ducts Pancreatic duct is blocked so enzymes don't reach duodenum and digestion is incomplete/impaired Large apetite Congestion and difficulty in breathing due to mucus in lungs Increased likelihood of lung infection
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Describe how adenoviruses are used to treat CF in the lungs
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Healthy CFTR gene removed from donor cell of a person who does not have CF The gene is cut out using restriction enzymes Viruses are grown in a sample of epithelial cells Plasmids extracted and cut using same restriction enzymes to produce the matching sticky ends DNA ligase used to splice DNA fragment to plasmid Plasmids taken up by adenoviruses and gene becomes part of viral DNA Viruses isolated and purified and then sprayed/inhaled into lungs Viral DNA passed to epithelial cells' nuclei
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What are the advantages and disadvantages of using adenoviruses to treat CF?
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+ Provides treatment of disease/relieves symptoms without genetic screening of embryoes - Immune system can develop resistance/create antibodies for the viruses so the virus type must be changed often for treatment - Delivery in inefficient, unsure if the allele is delivered and not all epithelial cells' DNA will be changed - Non-permanent fix - supplemented gene not passed onto the next generation - Virus may revert to a disease causing form
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Describe how liposomes with plasmid vectors are made and used to treat CF in the lungs
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Plasmids with the healthy CFTR allele are inserted into bacteria Bacteria multiply and produce many copies of the plasmid Plasmids extracted and coated in a sphere of phospholipids forming a liposome Sprayed into lungs via nasal/mouth spray/inhaler Thses liposomes can easily pass through the cell membranes of the epithelial cells in the lungs
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What is a vector?
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Plasmid which transfers genetic material into a cell from somewhere else (eg. a virus, another bacterium or a liposome)
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What are some advantages of gene therapy?
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Treatment of genetic diseases/relief of symptoms Ability to fully cure a genetic disease
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What are some disadvantages of gene therapy?
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Belief that screening is an invasion of privacy Possible increase in number of abortions and ethical issues associated with this Individuals known to be carriers/affected are put in higher risk groups for insurance making insurance difficult to get/very expensive Regulation of therapy so that it is only used to treat diseases and not used to modify/choose characteristics in a child ("designer babies")
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Define: Recombinant DNA
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DNA which results from the combination of fragments from two different organisms/when foreign DNA is incorporated into the plasmid from a bacterium
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Define: Restriction endonuclease
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Enzymes which cut DNA molecules between specific base sequences
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State some of the applications of genetic engineering
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Transfer of genes into bacteria to produce useful enzymes or proteins like insulin Transfer of new genes into crops and livestock so they acquire beneficial traits (eg Disease resistance) Transfer of genes into humans to cure genetic diseases
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What are the main stages in gene transfer, as used in genetic therapy
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Isolation of the specific DNA fragment/gene Insertion of the foreign DNA fragment/gene into a vector Insertion of vector into suitable hosts for replication Identification of the cells that took up the gene by use of genetic markers Growth of the host cells Extraction of the vectors containing the specific gene
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What are the two main ways of isolating a gene?
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Using reverse transcriptase Using restriction endonuclease
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Describe how reverse transcriptase can be used to isolate a gene
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Selected gene is identified using information from HGP mRNA is located using primer chemicals mRNA is extracted from a cell that uses the gene a lot, ie. produces large amounts of the protein coded for by the gene mRNA converted into cDNA by reverse transcriptase Complementary strand produced using DNA polymerase to produce a double helix DNA strand DNA strand incorporated into a plasmid
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Describe how restriction endonucleases are used to isolate a gene and insert it into a vector
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Restriction endonuclease cuts the "foreign" DNA at a recognition site Unpaired cut bases at the ends of the cut form sticky ends This process is called cleavage The same restriction enzyme is used to produce the same sticky ends in the plasmid DNA DNA ligase splices the foreign fragment into the plasmid (anneals the sticky ends) forming recombinant DNA
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Define: Reverse transcriptase
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An enzyme that converts a molecule of mRNA into a complementary single strand of DNA
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How are plasmids, used as vectors, removed from bacteria
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Bacteria are treated with alcohol to dissolve the cell membranes and other chemicals to dissolve cell walls Plasmids are separated from the cell debris
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What is significant about the plasmids used as vectors?
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They must contain at least one gene for the resistance to an antibiotic and (for use as a genetic marker) a second gene for resistance to a second antibiotic which is altered when the foreign fragment is incorporated
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Describe how a group of only the bacteria containing plasmids with a foreign DNA fragment is produced once a supply of vectors with the foreign fragment have been produced
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Plasmids are mixed with the bacteria Only a very small proportion of bacteria take up the plasmid The bacteria are all treated with an antibiotic Only the bacteria that took up the plasmid have resistance to this antibiotic, only these survive Genetic markers are used to separate the bacteria who took up the plasmids which had the foreign DNA and bacteria who took up the plasmids which did not have foreign DNA
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How are foreign DNA segments incorporated into vector plasmids and subsequently into bacteria for replication?
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By using the same restriction enzymes to cut the plasmid Mixing the fragments and plasmids along with DNA ligase Not all of the plasmids take up the desired fragment, some take up different foreign fragments present due to contamination Bacteria sample and plasmids mixed Only a very small percentage take up the plasmids
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State four genetic markers used to differentiate between bacteria containing vector plasmids with the desired foreign DNA segment and vector plasmids without the desired foreign DNA segment
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The desired DNA fragment may be radioactive The desired DNA fragment may disrupt a gene, in the plasmid, that codes for an enzyme that is detectable/produces a detectable change (eg makes the cell blue) The desired DNA fragment may disrupt a gene, in the plasmid, coding for a fluorescent protein, in the plasmid The desired DNA fragment may disrupt a gene, in the plasmid, that codes for the resistance to a second antibiotic
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Describe how a group of only the bacteria containing plasmids with the desired DNA fragment is produced, BY USE OF ANTIBIOTICS, from a group of bacteria some of which took up the plasmids with the desired gene and some of which took up the plasmids without the desired gene
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The desired foreign DNA fragment may disrupt a gene, in the plasmid, that codes for the resistance to a second antibiotic The plate with the bacteria on is replicated Replica plate is treated with the second antibiotic The cell colonies killed by the antiobiotic are the desired bacteria These colonies of bacteria on the original plate are extracted and cultured using fermentors
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State some advantages of using recombinant DNA technology
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Quantity production of complex proteins or peptides which cannot be made by other methods Removal of the need to use extracts from mammalian organs Uses in the production of higher yield crops with superior traits Health benefits in treating genetic diseases
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State some disadvantages of using recombinant DNA technology
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Concerns over its misuse Technically complicated and therefore very expensive on an industrial scale Difficulties in identifying the genes of value in a large genome Synthesis of the required protein may involve several genes each coding for a single polypeptide Treatment of DNA with restriction enzymes produces millions of fragments which are of no use Not all eukaryote genes will express themselves in prokaryrote cells
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State some potential hazards of using recombinant DNA technology
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Bacteria can readily exchange genetic material - this recombinant DNA may get into other organisms Deliberate use of anitbiotic resistant genes in E.coli which lives in the human gut means that these genes could be accidentally transferred to human pathogens Possibility of transfer of DNA with linked pathogenic genes (eg oncogenes that increase risk of caner) A new gene, on insertion, may disrupt a normal gene function - could lead to development of new pathogens
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What is so dangerous about bacteria being able to readily exchange genetic material?
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This recombinant DNA may get into other organisms Could lead to herbicide resistant weeds
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What are the advantages of treating CF with gene therapy as opposed to using non-genetic treatments?
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Gene therapy is more effective and reduces/treats the symptoms (more/better) Reduces the number of injections and antibiotics needed to be taken by the patient Removes the need for dietary restrictions on patient Less intensive monitoring of patient needed
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Describe arguments for and against the genetic screening of normal individuals for genetic disorders
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+ Prevents disease causing alleles being passed on + Reduces the incidence of the gene in the population - Increased anxiety in patients who may not develop the disease - Which can affect lifestyle choices - Confidentiality issues - Insurance companies may refuse cover to individuals with disease causing alleles
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Why must the same restriction enzyme be used to cut the vector plasmid and the chromosomal DNA when forming recombinant DNA?
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To give matching sticky ends so that the foreign DNA fragment can be joined to the vector DNA by complementary base pairing
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What is the function of DNA ligase in gene therapy?
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Splice sticky ends together and thus join the foreign DNA fragment into the vector DNA
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What events must occur once a normal CFTR gene is inserted into a lung epithelial cell of a patient with CF for the treatment to work?
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Gene gets into the nucleus Gene is incorporated/inserted into chromosome Transcription of gene and translation of resulting mRNA to give rise to the functional protein
