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DNA microinjection is a pioneering technique that involves the direct injection of a gene construct into the pronucleus of a fertilized ovum, enabling genetic manipulation in mammals. This method, effective since the early 1980s, allows for the introduction of genes from the same or different species. While it provides opportunities for transgenesis, the random nature of DNA insertion can lead to gene expression variability. The process includes superovulation of female mice, followed by microinjection of fertilized eggs and transferring them into a foster mother for successful development.
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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
