Improving the generation of genomic-type transgenic mice by ICSI

Transgenes included in genomic-type constructs, such as yeast artificial chromosomes (YAC), P1-derived artificial chromosomes, or bacterial artificial chromosomes (BAC), are normally correctly expressed, according to the endogenous expression pattern of the homologous locus, because their large size usually ensures the inclusion of all regulatory elements required for proper gene expression. The use of these large genomic-type transgenes is therefore the method of choice to overcome most position effects, commonly associated with standard-type transgenes, and to guarantee the faithful transgene expression. However, in spite of the different methods available, including pronuclear microinjection and the use of embryonic stem cells as vehicles for genomic transgenes, the generation of transgenic animals with BACs and, particularly, with YACs can be demanding, because of the low efficiencies requiring extensive microinjection sessions and/or higher number of oocytes. Recently, we have explored the use of intracytoplasmic sperm injection (ICSI) into metaphase II oocytes as an alternative method for the generation of YAC transgenic mice. Our results suggest that the use of transgenic strategies based on ICSI significantly enhances the efficiency of YAC transgenesis by at least one order of magnitude.