Microinjection method: In another approach fertilised eggs are microinjected by DNA. Microinjection requires excess of fertilized eggs. Gene transfer by microinjection is the predominant method used to produce transgenic farm animals. Since the insertion of DNA results in a random process, transgenic animals are mated to ensure that their offspring acquire the desired transgene. However, the success rate of producing transgenic animals individually by these methods is very low and it may be more efficient to use cloning techniques to increase their numbers. For example, gene transfer studies revealed that only 0.6% of transgenic pigs were born with a desired gene after 7,000 eggs were injected with a specific transgene.
To obtain good number of eggs, donor female is induced to superovulate and fertilised eggs are obtained after mated with male. In this method, the novel gene in an appropriate copy number is directly introduced into the male pro-nucleus of a fertilised egg just before its fusion with female pro-nucleus. Male pronucleus is clearly visible as it is larger than the female pronucleus in the fertilised egg. The fertilised egg carrying transgenes are implanted into receptive female (foster mother). Implanted females or foster mother gives birth to progenies carrying transgene. Finally the animals which carry transgenes are identified and bred to establish transgenic lines. This method is still the widely accepted method in mouse and rabbit model, however, in farm animals, opaqueness of zona pellucida and lack of clear vision of male pronucleus are the main limitations. Further random integration of gene in host genome may require longer time to produce a pure line of transgenic livestock.