Identification of the Sex of Pre-implantation Mouse Embryos Using a Marked Y Chromosome and CRISPR/Cas9

Abstract

Although numerous attempts have been made to alter the sex ratio of the progeny of mammals, the limitations of current technologies have prevented their widespread use in farm animals. The presence or absence of a Y chromosome determines whether a mammalian embryo develops as a male or female, and non-invasive genetic reporters such as fluorescence protein markers have been intensively applied in a variety of fields of research. To develop a non-invasive and instantaneous method for advance determination of the sex of embryos, we developed a Y chromosome-linked eGFP mouse line that stably expresses green fluorescent protein under the control of the CAG promoter. The development of the CRISPR/Cas9 system has made it easy to deliver an exogenous gene to a specific locus of a genome, and linking a tracer to the Y chromosome has simplified the process of predicting the sex of embryos collected by mating a Y-Chr-eGFP transgenic male with a wild-type female. XY embryos appeared green, under a fluorescence microscope, and XX embryos did not. Y chromosome-linked genes were amplified by nested PCR to further confirm the accuracy of this method, and the simultaneous transplantation of green and non-green embryos into foster mothers indicated that 100% accuracy was achieved by this method. Thus, the Y-Chr-eGFP mouse line provides an expeditious and accurate approach for sexing pre-implantation embryos and can be efficiently used for the pre-selection of sex.

Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive

“…The generation of Y-linked fluorescent marker lines has been reported in mosquitoes, flies, and mice 25 , 41 , 42 . Here, we report the first generation of a transgenic line that robustly expresses the Cas9 endonuclease from the Y chromosome in insects.…”

Advances and challenges in genetic technologies to produce single-sex litters

“…Furthermore, previous studies have successfully generated in-frame knock-in Uty-eGFP reporter ESC lines [106]. Moreover, Zhao and colleagues recently generated a Y-linked reporter mouse line wherein eGFP expression was driven by a constitutive promoter and shown to be expressed in preimplantation embryos [113]. The transgene was inserted into an intergenic region between Uty and Ddx3y, thereby opening up new possibilities of Y-chromosome knock-in targets.…”

Y chromosome in health and diseases

“…Hence, TgTSPY9 is a humanized mouse model for TSPY gene. Accordingly, with the recent advances of genome editing and transgenic technologies [ 64 – 67 ], targeting the integration of human MSY gene(s) onto the mouse Y chromosome could be viable strategies to model and study their functions in biological processes, involved in various aspects of development, physiology and disease pathogeneses.…”