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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
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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
