Genetically modified animals have been used as models in broad range of studies including pharmaceutical biology. Designing and construction of transgene constructs are the first indispensable task in generating model animals. In addition to the classical restriction enzyme-based method, still holds some advantages in generating precise constructs, site-specific recombinase-based and homologous recombination-based DNA engineering strategies (e.g. Gateway and Red/ET recombineering, respectively) have been developed and widely used for vector construction or BAC modification. In this review, the three construction methods are described and their applications are discussed such as tandem assemblies of multiple components and modification of large DNA molecules. Combinational use of these E. coli-based recombinant DNA technologies enables the generation of precisely designed vectors useful for desired genome modification for future analyses.