Mouse - experiments

Question 1

Gene Trap

Answer 1

• Insertional mutagenesis with a suitable DNA vector as this allows for easy identification of the modified locus.
• The relevant constructs are called gene traps and contain a splice acceptor site, a reporter gene, and a selectable marker.
• The construct is transfected into ES cells
• It it integrates outside a gene it remains inactive because it has no promoter of its own
• If it integrates within gene it should function as the last exon, with the splic acceptor enabling encoporation into the coding sequence.
• Because the endogenous gene is dirupted by the insertion it is likely to be mutated to inactivity or reduced function.
• One important gene trap mouse strain is the Rosa26.
  
  • The trapped gene encodes an untranslated RNA of unknown function
  • It is ubiquitously expressed at all stages in all tissues.
  • If ubiquitous expression of a transgene is required it likely knock-in to the Rosa26 locus

Question 2

Enhancer Trap

Answer 2

• This is a gene trap that lack the splice acceptor and has its own minimal promoter instead
• It is inserted into the genome within the range of an endogenous promoter or enhancer this will complement the minimal promoter and enable significant transcription of the reporter gene
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