The injection of ES cells at an earlier time-point, i.e. the 8-cell-stage, leads to an F0 generation of mutant mice that is completely ES cell-derived and allows immediate phenotyping.
In 2007, Poueymirou et al. developed a laser technology to perforate the zona pellucida followes by injection of the ES cells into the perivitelline space (published in). This was a major technological advance to facilitate the injection of eight-cell-stage embryos.