Biotechnology and Bioinformatics (1+1)

2.2.5.1. Microinjection

Microinjection is the most common method of gene transfer . It involves the use of an injection pipette, the dimensions of which depend on the target species: e.g., pipettes of inner diameter of 3-5 µm are used for tilapia. Soon after fertilization the gene is microinjected into the cytoplasm since the egg nucleus is not visible in the fishes. The site of injection varies from species to species. In fish eggs, the nucleus or pronucleus cannot be seen using conventional light microscopy, mainly because of the opaqueness of the chorion and /or the cytoplasm. Hence, most transgenic fish studies have opted for cytoplasmic injection of DNA, following fertilization.

The target is the thin layer of ooplasm under the chorion or developing blastodisc. The injection pipette must penetrate the chorion (which is often thick and opaque, except in some species such as catfish and medaka, which have transparent and thin chorions) and the membrane of the fertilized egg.

Several methods of pretreatment have been reported, including a two-step method which involves

• piercing the chorion with a broken pipette before microinjection into its ooplasm, dechorionation and prevention of chorion hardening,
• microinjection through the micropyle and
• microinjection before hardening of the chorion.

The possibility of damaging eggs during microinjection is high and this technique requires a great deal of skill.

• The survival rates of different species of transgenic fish produced by this method have been reported to range from 5 to 90%.
• Linearised DNA rather than circular DNA is injected for the greater probability of the former to get integrated into the host’s genome .
• Higher amount of DNA is used for cytoplasmic gene transfer than when it is injected to the pronucleus.
• Some species has softer chorion such as catfish, Zebra fish.
• Small volume of the solution 1-2 nl of DNA containing >10⁷ copies should be injected.
• The rate of survival and integration of the transgene after microinjection varies widely in different species of fishes and in different batches of the same species.
• Although microinjection is time consuming, laborious, species-specific and technically demanding, it remains the most widely used method for gene transfer in fish.