Fig. 2.

Storyboard of gene-targeted manipulation. Before the complete sequencing of animal genomes, investigators wishing to target specific genes for knockout in animal models were required to initially map genomic DNA to identify the exons, introns, and unique restriction sites within the gene of interest. Steps involved include 1) endonuclease digestion of genomic DNA; 2) creation of a phage DNA library; 3) screening of phage library; 4) Southern blot analysis with phage DNA probes; 5) rescue of cDNA fragments into bacterial plasmids; 6) sequencing; and 7) design and construction of a targeting vector with upstream and downstream arms of DNA to promote homologous recombination.