Paediatrics (RCH) - Theses

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    Structural and Regulatory Changes to the Mouse Y Chromosome
    Robinson, Jessica Louise ( 2020)
    The X chromosome and the Y chromosome are the sex chromosomes of mammals. While the X chromosome carries many genes with functions beyond sex, the Y chromosome is relatively gene poor. It serves as the trigger for development of a male embryo, with its genes having roles in testis determination and function. The sex chromosomes exist as a pair; in females, this pair are XX, and in males, XY. Two different mouse Y chromosomes were the focus of this project. The first was a transgenic fluorescent reporter Y chromosome, Ddx3y^mKate2, which was created to be used as a tool in chromosome instability research. The second was the naturally occurring Y chromosome from the A/HeJ inbred mouse strain, Y^A/HeJ. The work associated with these two Y chromosomes comprised the two studies in this project. The Ddx3y^mKate2 Y chromosome was generated by targeted insertion of a transgene cassette containing the red fluorescent protein gene, mKate2, into the mouse Y chromosome adjacent to the Ddx3y locus. This was selected as the relatively broad expression of Ddx3y indicated a permissive environment for expression of the mKate2 transgene. As male mice are XY, all male Ddx3y^mKate2 mice carried the fluorescent reporter. The mKate2 fluorescence in this strain has been previously assessed, revealing that the reporter was detectable from the preimplantation embryo, through to sexual maturity. To finalise the characterisation of the Ddx3y^mKate2 mouse, this study assessed the fluorescence in the Ddx3y^mKate2 male in advanced age. Additionally, this study characterised another transgenic reporter strain, Hprt^DsRed-Express, to serve as a comparison. To demonstrate the utility of the Ddx3y^mKate2 reporter, the Ddx3y^mKate2 Y chromosome was crossed onto two known chromosome instability backgrounds: the Trp53 knockout and Cenpagfp fusion knock-in backgrounds. Following in vivo and in vitro experiments, it was determined that the Cenpagfp background was the better background to model the Ddx3y^mKate2 reporter Y chromosome; however, more work using this genetic background in embryonic stem cells is advised for assessing the Ddx3y^mKate2 reporter Y chromosome. The Y^A/HeJ chromosome from the inbred A/HeJ strain was causally associated with a disturbance in the testis determination pathway. The Y^A/HeJ chromosome resulted in a subset of male mice having developed either ovotestes (gonads containing ovarian and testicular tissue) or abnormally small testes without epididymal sperm. It was confirmed that the Y^A/HeJ chromosome still carried the testis triggering gene, Sry. The gonadal and cytological abnormalities associated with this Y chromosome lead to the speculation that there was a structural change at or near its centromere. This study extended the characterisation of the A/HeJ phenotype, including identification of an age-related decline in male gonad mass, as well as assessment of the Y^A/HeJ chromosome. It was confirmed that the structural change to the Y^A/HeJ chromosome was a halving of the Rbmy tandem repeat array. This was predicted to adversely affect Sry gene expression during testis determination, resulting in the A/HeJ gonadal phenotype. Assessment of Sry regulation and gene expression from the Y^A/HeJ chromosome is recommended to complete the work associated with this Y chromosome.