Lecture22 &23 DNA microinjection

DNA MICROINJECTION direct microinjection of a chosen gene construct 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 (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 into the pronucleus of a fertilized ovum one of the first methods that proved to be effective in mammals (Gordon and Ruddle, 1981) 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 introduced DNA may lead to the over- or under-expression of certain genes

The insertion of DNA is random process The insertion of DNA is random process high probability that the introduced gene will not insert itself into a site on the host DNA 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 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 induced to act as a recipient by mating with a vasectomized male Applicable to a wide variety of species. Applicable to a wide variety of species.

Procedure for DNA microinjection fertilized eggs to be inoculated by microinjection is increased by superovulation 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. 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 super- ovulated females are mated and killed the fertilized eggs are flushed from their oviducts the fertilized eggs are flushed from their oviducts microinjection of the fertilized eggs microinjection of the fertilized eggs the microinjected transgene construct is often in a linear form and free of prokaryotic vector DNA sequences the microinjected transgene construct is often in a linear form and free of prokaryotic vector DNA sequences

Transgenic mice by DNA microinjection