Microinjection - In this method, DNA solution is injected directly into the nucleus of a cell or into the male pronucleus of a fertilized one- to two cell ovum. Typically, a microinjection assembly consists of a low power sterioscopic dissecting microscope (to view the ovum and the entire process) and two micro­manipulators, one for a glass micropipette to hold the ovum by partial suction and the other for a glass injection needle to introduce the DNA into the male pronucleus.

The male pronucleus is much larger than the female pronucleus of fertilized mammalian ova. However in fish ova, the DNA is injected into the egg cytoplasm.

In mice, the microinjected embryos are cultured in vitro upto the morula or blastocyst stage. The surviving embryos are then transferred into the uterus of surrogate mothers, i.e., females, which have been made receptive by hormone treatments; these embryos develop to full term and give rise to progeny mice.

A proportion of the progeny so produced will be transgenic in that all their cells will contain the transgene stably integrated into their genomes. In mice, an average of about 3-6% of the progeny derived from micro injected embryos are transgenic; the frequency is much lower in other animals, e.g., < 1 % in sheep and pigs.

In case of fish about 35-80% of the embryos survive microinjection, of which 10-70% may be transgenic. The transgenic animals contain the transgene in their germ cells and, as a consequence, pass it on to their progeny; transgenes show typical Mendelian inheritance.

Subsequently, trans gene integration occurs only in some cells of the embryo; this results in chimaeric progeny. Pure transgenic individuals can be recovered through suitable breeding schemes from such chimaeric animals in whose germ cells the transgene has become suitably integrated.

All the transfection techniques are applicable to cultured animal cells, but microinjection is ordinarily not used due to the tediousness of the technique and the limited number of cells that can be handled.

For transfection of mice embryos, the preferred techniques are microinjection, retroviral infection and embryonic stem cell technology, microinjection being the most commonly used. Embryos of other animals are generally transfected by microinjection technique only. In case of fish embryos, microinjection and electroporation are routinely employed.