1. Transgene DNA Construct: cDNA of a gene of interest along with the regulatory elements (promoter, terminator etc) are used to design the DNA construct. Promoter and 3’ UTR are necessary for proper gene regulation.  Some evidence shows that intron splicing plays role in gene expression in mammalian system. Hence introns are also included in the construct sometimes.  Construct should be linearized before injection.

2. Embryo Collection: Donor parental strain for the production of embryo are selected considering factors like response to super ovulation (ability to produce mature ova at large number), frequencies of embryo survival following microinjection, size of pronuclei and incidence of specific pathologies inherent to strain.

Successful super ovulation depends on the strain, age and weight of the animal. Breeding should be monogamous. After post-mating, embryos are collected at single cell stage and proceed for microinjection.

3. DNA Microinjection:  The purified double stranded linear DNA construct containing the transgene DNA sequence is introduced to host cell by microinjection. The foreign DNA must be integrated (although random) into the host genome prior to cell division. To facilitate this transgene DNA is introduced into zygote at the earliest possible stage (pronuclear period) immediately after fertilization. Usually male pronucleus is preferred because it is larger and easier to inject. The host chromosome at the site of integration generally undergoes duplication, deletion or rearrangements due to transgene incorporation. This may lead to insertional mutagenesis and thus producing detectable phenotypic trait.
 
Note: Critical points for successful DNA microinjection technique-

• Careful collection of relatively large group of accurately single cell embryo.
• Embryo transfer to suitable recipient female (standardized in each case).
• Construction and preparation of transgene DNA fragment to be injected.