The role of the Boris gene in generation of methylation imprints during mouse spermatogenesis

Brother of the Regulator of Imprinted Sites (BORIS; CTCFL) is a gene that has been implicated in the expression of genetically imprinted genes. In adult animals, it is normally expressed in the testes and is reactivated and expressed in many cancer cells and embryonic stem cells. However, it is well known that the appropriate gene expression is crucial for the fertilization and further fetal development. Therefore, we genetically engineered mice in such a way to allow expression of the Boris gene to determine its effects on spermatogenesis, male fertility (both in vivo and in vitro), fetal growth and viability and also the formation of methylation imprints in H19/Igf2 locus.The mouse Boris gene, two different forms as mutant and normal, was cloned into the Bidirectional Tet plasmid (pTRE-Tight-Bl-AcGFP1) that contains GFP and a tetracycline (Tet) responsive element which permits expression of the Boris transgene when doxycycline is present in the drinking water. This plasmid was injected into mouse zygotes to create the transgenic strains. Mice born from injected zygotes were screened by PCR to determine founder lines. These mice were then bred to transgenic strains that carry testis specific promoters, Protamine Cre or Synaptonemal complex Cre, and tetracycline controlled transactivator protein (rtTA) for the ectopic gene induction. The triple transgenic mice were then administered of a 2-3 month course of doxycycline (2mg/ml supplemented with %5 sucrose) in drinking water. RNA samples were extracted from testis and quantitative real time PCR method (qRT-PCR) was performed. Testis samples were further evaluated by light and fluorescein microscopy and proliferative index (PCNA) and percentage of apoptotic cells were calculated. Females bred to such males and who were on doxycycline during the pregnancy were allowed to give birth and altered embryonic defects in the offspring were analyzed. Bisulfite sequencing was performed to analyze H19 DMD methylation status.Our results showed that both Boris transgenic mice were fertile but there was a potential relation between Boris and spermatogenesis. The offspring from doxycycline fed mutant and normal Boris transgenic mice were smaller with a higher postnatal mortality rate compared to that of control mice. The affected pups, confirmed by genotyping, showed retarded growth in most organs. In addition, H19 DMD methylation status was found to be significantly different than controls with the adult and offspring DNA samples.Our study is of importance for being the first to investigate the possible relationship between BORIS and embryogenesis defects and fetal development. Future work will focus on possible mechanisms for how the Boris gene, when aberrantly expressed during embryogenesis could alter normal development.