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Transcript
Lecture22 &23
DNA microinjection
DNA MICROINJECTION
• direct microinjection of a chosen gene construct
• (a single gene or a combination of genes) from another member of
the same species or from a different species
• into the pronucleus of a fertilized ovum
• one of the first methods that proved to be effective in mammals
(Gordon and Ruddle, 1981)
• the introduced DNA may lead to the over- or under-expression of
certain genes
• The insertion of DNA is random process
• high probability that the introduced gene will not insert itself into a
site on the host DNA
• manipulated fertilized ovum is transferred into the oviduct of a
recipient female or foster mother
• induced to act as a recipient by mating with a vasectomized male
• Applicable to a wide variety of species.
Procedure for DNA microinjection
• fertilized eggs to be inoculated by microinjection is increased by
superovulation
• female mice are given an initial injection of pregnant mare’s serum and
another injection, about 48 hours later, of human chorionic gonadotropin.
• the super- ovulated females are mated and killed
• the fertilized eggs are flushed from their oviducts
• microinjection of the fertilized eggs
• the microinjected transgene construct is often in a linear form and free of
prokaryotic vector DNA sequences
Transgenic mice by DNA microinjection