Genetic engineering

• How to create transgenic mouse

  1. Inject forein DNA into Pronuclei (using microinjection)
  2. Egg transferred to foster mother
  3. Analyse offspring for presence transgene (PCR) - to see if they contain transgene
  4. Breed positive mice to generate homozygous mice
• Use of transgenic animals

  “Reporter” genes to follow gene expression

  Models of disease

  Altered characteristics – growth rate, meat/milk yield, milk quality

  “Pharming” – production of pharmaceuticals in milk

  Insect control

  General interest/fun - GloFish®
• Use of reporter gene (chondro-mouse)

  Green Fluorescent Protein under control of the collagen II promoter.

  Green fluorescence where collagen II is being made, expressed
• What causes spinocerebellar ataxia?

  Abnorma; ataxin protein which is neurotoxi
• Human growth hormone expressed in a transgenic mouse model of acromegaly.
• What organism can be used to produce human antithrombin

  Goats
• genetically engineered insects:

  Sterile male ades aegypti - due to high levels of radiation (didn't work so use genetically engineered to produce only male)

  Out compete fertile males causing population

  Used to deal with dengue fever
• Issue with Multicoloured Glo fish

  no benefit to society
• What is a knockout mouse

  Wild type of gene is replaced with an inactive gene to create a knockout

  Used to study what a gene does
• What is the difference between transgenic and knockout mouse

  Transgenic: Insertion of exogenous DNA into random

  non-homologous site in mouse genome.

  Frequency high so relatively easy to produce such mice.

  Knockout: Insertion of modified DNA at a specific

  homologous site in mouse genome.

  Frequency very low so difficult to produce such mice.
• How to creat knockout mouse

  Embryo stem cell,

  Blastocyst grown in supplemental media (Sterile conditions)

  Normal gene is changes in to a replacement construct

  Replacement construct: Neomycin resistance = marker of homologous recombination, THymidine kinase= non-homologous marker
• Homologous recombination:

  Crossing over occurs at right place
• Nonhomologous:


  ???
• Positive and negative selection:

  Treat with neomycin (positive selection)

  Treat with ganciclovir (negative selection)
• Creation of knockout mice:

  Use two types of mice (one black, one white here). Use ES cells from black mice to generate selected cells and inject back into early embryos from white mice.

  Offspring are all white or chimeric (both black and white).

  Mate chimeric with white to produce small number of all black offspring.

  Interbreed black mice to make homozygous knockout mouse.
• What are the uses of knockout mice

  • Test gene function:
  • Models of human diseases:

  –CFTR– cystic fibrosis

  –Dystrophin – Duchenne muscular dystrophy

  –Rb gene – retinoblastoma

  –globin - thalassaemia
• GENE THERAPY:

  Treating diseases with genes - in adult:

  A mutant gene is replaced, or supplemented, by a normal one

  OR

  Therapeutic gene is introduced -correct sequence (dominant) (e.g. anti-cancer gene)

  in vivo or ex-vivo approaches
• Example of targets for gene therapy

  Single gene disorders:

  –Haemophilia

  –Cystic Fibrosis

  –Duchenne muscular dystrophy

  –Sickle cell anaemia

  Cancer

  Rheumatoid arthritis

  Erythropoietin for kidney patients
• Early attempts of gene therapy

  1. Targeted Cystic Fibrosis – in vivo approach
  2. Added wild type cystic fibrosis gene to adenovirus vector
  3. Squirted aerosol containing virus into lungs of cystic fibrosis patients
  4. For 8 weeks - improvement in symptoms
  5. At around 8 weeks, host immune system attacked cells carrying virus and wiped them out - removing normal cystic fibrosis gene and any chance of cure
• Problems of adenovirus as vector:

  Is a common cold virus - we have antibodies against it

  May elicit severe immune response

  Inserts randomly into genome, may cause worse disease

  Only inserts into chromosomes if the host cell actively replicating DNA
• Adeni-associated virus:

  Very small - doesn’t elicit much immune response

  Infects both non-dividing and dividing cells

  Vectors developed to control protein expression
• Severe combined immunodeficiency disease: SCID is a rare inherited disorder that results in the absence of all or most of the immune system
• Stem cell gene therapy:

  1. insert RNA verison of normal allele into retrovirus
  2. Virus infefct bone marrow cells that have been removed from the patient and culture
  3. Viral DNA carrying the normal allele insert into chromosomes

  May lead to leukaemia
• CRISPR - Cas9:

  CRISPR -  clustered regularly interspaced short palindromic repeats

  Cas9 – CRISPR-associated; the “genetic scissors” portion of CRISPR/Cas9

  Cas9 = enzyme that chops up DNA but need CRISPR

  BACTERIAL immune response: chops up viral DNA and inserts into genome, which allows to cut up further viral infections
• Cas9:

  Is given a guide RNA to match sequence in faulty or abnormal gene,

  This allows a double-stranded cut within target region

  Cas9 cuts around faulty gene, this either leads to cell repair by two ways non-homologous end joing OR homology directed repair (WE want this one)