Pronuclear injection

Question 1

Steps involved in pronuclear injection transgenics

Answer 1

• 4 week old females are super ovulated and mated
• Fertilised eggs are collected and allowed to mature until the male pronuclei starts to move to the centre of the zygote
• DNA is microinjected into the male pronucleus
  
  • 200-400 eggs/day
  • 75% survival
• Embryos are transferred into the oviduct of pseudo pregnant females
• 18 days later pups are born
  
  • 25% success rate
• Screen DNA to identify transgene incorporation
• 10% genes are transgene positive

Question 2

What are the uses of pronuclear injection transgenics?

Answer 2

• For disease models, most commonly:
  
  • Trans-speciation
  • e.g. human gene in mouse
• Repression of a gene
  
  • shRNAi inhibition of expression
• Over expression of a gene
• Normal expression - its own promoter
• Ectopically expression - another promoter
• Different cell type, different time, and Cre
• Reporter of gene expression
• Promoter or non-coding region analysis
• Pronuclear injection also used in CRISPR genome editing

Question 3

What must a transgene be?

Answer 3

A transgene must be a complete transcriptional unit

Question 4

What does a complete transcriptional unit contain?

Answer 4

• Regulatory regions
  
  • Enhancer region
    
    • To direct high levels of expression
  • Promoter region
    
    • That contains contain the basic transcriptional machinery and start sight
  • Gene
  • Start and stop codon
  • 5’ and 3’UTR
  • 3’ end of gene will have intron
    
    • This is to stop transcription
    • PolyA tail to stop transcription

Question 5

LOOK AT NOTES FOR EXAMPLES

Answer 5

LOOK AT NOTES FOR EXAMPLES