In vivo gene manipulation and gene expressioncontrol techniques

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In vivo gene manipulation and gene
expression-control techniques

• Knock-outs, Transgenics, Knock-ins
• Simon Hunt
• Dunn School of Pathology
• email simon.hunt_at_path.ox.ac.uk
• You are welcome to use the material in this
  lecture for your own private study further
  distribution is not allowed. Available by link
  from http//www.psb.ox.ac.uk/Newfhs/09imm.htm

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Inserting/replacing genes

• Always start with cloned DNA containing all or
  part of the gene of interest
• Usually the coding region derived from cDNA, or
  the upstream promoter region
• If this gene is to be expressed it must itself
  have a suitable promoter
• Hence can be engineered to be expressed
  tissue-specifically
• (and in the wrong tissue if desired ectopic
  expression)
• Transgenesis
• Gain of DNA, equivalent to Transformation by DNA
  in vitro
• A shotgun by microinjection of lots of cloned
  DNA to pronucleus of fertilised egg , hoping that
  some of the construct will incorporate somewhere
• Many failures needs patience, many and a
  good screening method

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Transgenesis protocol
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Blastocyst injection a steady hand

• Inoculation by microneedle to fertilised egg
• Repeat dozens of times

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Success may sometimes be obvious
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Making transgenic (Tg) mice
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Knockouts using Embryonic Stem cell line

• ES cells Embryonic Stem cell line truly
  totipotential, from mouse teratomas
• Limited no. of mouse strains

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Targeted homologous recombination
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Specific interference with gene expression
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Recommended reading

• Website http//cancer.ucsd.edu/tgm/genetargeting.
  asp covers all aspects, including conditional
  gene targetting
• Article Rabbitts, T. H. and M. R. Stocks (2003).
  "Chromosomal translocation products engender new
  intracellular therapeutic technologies." Nature
  Medicine 9(4) 383-6. http//dx.doi.org/doi10.103
  8/nm0403-383 Survey of gene alteration methods,
  including gene expression controls that may be
  possible in future