Generation of the Y-chromosome linked red fluorescent protein transgenic mouse model and sexing at the preimplantation stage

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.