GM animals

• Transgenicanimals

  They have additional genes [known as transgenes] which are not normally expressed.
• Creation of transgenic mice

  
  

  1. Use microinjection to insert foreign DNA into the male pronuclei

  3. Eggs transferred into foster mother

  4. Analyse offspring for presence of transgene using PCR

  5. Breed positive mice to generate homozygous mice
• Describe how transgenic mouse can be used with a reporter gene

  A green fluorescent protein acts as a reporter protein and is under the control of the collagen II promoter. The green fluorescent protein is expressed whenever collagen II is and this allows for the expression of collagen II to be studied during development.
• What is spinocerebeelal ataxia

  This condition impairs motor function. It is caused by a transgene which codes for an abnormal ataxin protein which is neurotoxic.
• How are altered characterisitcs used

  They are used to analyze the phenotypes and understand the effects of a gene.
• "Pharm" animals

  This is the production of pharmaceutical products using animals
• Describe the use of goat as a pharm animal

  Human antithrombin is produced in goat's milk as a treatment for blood pressure disorders.
• Describe the use of GM mosquitoes for dengue fever

  The mosquitoes are a vector for dengue fever.

  The males are irradiated to make them sterile this produces a population crash.
• Knockout

  Replacement of a wild type gene with an inactive gene. The inactive gene is a mutated version of the wild type which is unable to function or functions incorrectly
• Differences between transgenic and knockout mice

  • Transgenic: Insertion of exogenous DNA into random non-homologous site in mouse genome, frequency high
  • Knockout: Insertion of modified DNA at a specific homologous site in mouse genome, frequency very low
• Creation of knockout mice

  Insertion of construct into embryonic stem cells.

  Select the homologous mutants.

  Fur colours is used as a reporter gene to show the passage of the construct through the offspring.

  Black [dominant gene] mice contain the mutated construct and the embryos are inserted into the white [recessive gene].

  Incomplete dominance produces either white or chimeric offspring.

  Mate chimeric with white to produce black offspring.

  interbreed the black mice to produce homozygous knockout mice.
• Uses of knockout mice

  • Test gene function
  • Models of human diseases caused by a single gene mutation
• How is a knockout gene made

  The replacement construct is similar in structure to the normal gene.

  One of the exons is replaced by a neomycin resistance gene.

  And a thymidine kinase gene is placed upstream of the gene.

  The neomycin resistance and thymidine kinase genes act as selection markers
• How is neomycin resistance used as a selection marker

  This is positive selection and used to remove the cells which did not take up the transgene.
• How is thymidine kinase used as a selection marker

  This is negative selection. The thymidine kinase gene makes cells susceptible to ganciclovir. This kills the cells that have taken up both the marker genes.
• Describe how ES cells are induced to carry the knockout gene

  Homologous recombination in which identical segments cross over. This results in the construct being inserted without the thymidine kinase gene. These are said to be the mutated cells. They are resistant to both neomycin and ganciclovir.

  
  

  Nonhomologous recombination in which sequences outside of the gene have homology to the ES cells DNA. These sequences undergo recombination and the construct is inserted with the thymidine kinase gene. These cells are resistant to neomycin but susceptible to ganciclovir. H
• What are the steps in selecting for knockout genes

  Positive selection with neomycin removes the non-mutated cells.

  Negative selection with ganciclovir removes the nonhomologous mutants.
• What is gene therapy

  The treatment of disease with genes.

  This can be by replacing the mutant gene or adding an additional wild type for the gene.
• What are the targets for gene therapy

  Single gene disorders and cancer.
• Describe the early attempts at gene therapy for CF

  This was an in vivo technique.

  The wild type cystic fibrosis gene was added to an adenovirus vector which was sprayed into the lungs.

  The downside was that the host immune system attached the cells carrying the virus.
• What are the problens of using an adenivurs as a vector

  It may elicit a severe immune response and most people have antibodies against it.

  It inserts randomly into the genome and may cause severe disease.

  Only inserts when the host is replicating DNA.
• What does the adenovirus cause

  The common cold
• Describe the benefits of using an adeno-associated virus

  It is small and so doesnt causse an immune response.

  It is able to infect both dividing and non-dividing cells.
• What is SCID

  Severe combined immunodeficiency disease
• Describe the use of stem cell therapy for SCID

  This is an in vitro technique.

  The RNA version of the wild type allele was inserted into a retrovirus.

  The retrovirus infects bone marrow cells that have been cultured.

  The viral DNA inserts into the chromosome.

  These cells are inserted into the patient.