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Non-Mendelian inheritance of the t-haplotype from heterozygous male mice to their offspring is also known as transmission ratio distortion.
The t-haplotype, a variant form of the t-complex, spans nearly one third of mouse chromosome 17 and encodes several distorter genes and one responder gene, which lead to formation of two different sperm populations during spermatogenesis.
Early expressed distorters activate signalling cascades of the sperm motility kinase, smok1, resulting in a negative effect on the motility of all sperm, while the responder counterbalances this situation only in sperm carrying the responder gene.
This gives these so-called t-sperm an advantage during fertilisation, and leads to an abnormally high inheritance. Previous work has demonstrated that a responder-based transgene shows haploid-specific transcript expression, which means that the mRNA is restricted to only half of all round spermatids, although these cells are connected via cellular bridges and thus exchange of gene products can occur.
Furthermore, the responder protein is detectable only in the flagellum of elongated sperm, which means its translation takes place during late stages of spermatogenesis.
This shows that translational regulation might be involved in responder gene expression and in silico analysis revealed already possible regulatory elements for translational control of the responder within the 5’-untranslated region (5’UTR) of the responder transcript. In the context of this work different responder-based transgenes were cloned to generate transgenic mice and to investigate the haploid specificity of the t-complex responder in vivo on transcript and protein level by using in situ hybridisation and immunohistochemistry and functionally in a transmission ratio distortion test.
In order to address the importance of regulatory elements of the 5’-UTR in responder expression several deletion constructs were generated and expression analyses in vivo revealed that the 5’-UTR was indeed involved in transcript localisation.
When the same 5’-UTR deletions were examined in combination with the responder coding sequence, it was demonstrated that the coding region was necessary for transcript stability and successful translation, along with at least the last third of the 5’-UTR region. Additionally, analysis of specific subdomains within the coding region of the responder using deletion constructs within this region, revealed that successful translation of this gene was dependent on the presence of at least the regulatory subdomain in the coding sequence. In order to functionally verify observations from the deletion studies, transgenic mice harbouring the coding region deletions were used in a transmission ratio distortion test, which measured the rate of transgene inheritance in the presence of distorters. In terms of transmission ratio distortion this test was inconclusive, and was thus insufficient to answer the question of which responder subdomain has adequate responder activity. The test further revealed possible shortfalls in the transgene integration strategy chosen. A strategy using recombinase-mediated cassette exchange of transgenic constructs integrated as a single copy in the ColA1 locus was used to generate mice, but within this work the locus itself revealed some unexpected positional effects resulting in low or completely absent expression, thereby impacting the outcome of in situ hybridisation analyses, immunohistochemistry, and of the transmission test. The intention of this work was to identify regulatory elements in the responder transcript which are important for the special expression of this gene and which might be used to manipulate other genes in the mouse or in other species.
This work demonstrated that the 5’-UTR of the responder is involved in transcript localisation while the coding region only in combination with the 5’-UTR is controlling transcript stability and translation. Therefore the results of this work revealed that post-transcriptional regulation of the responder is quite complex and depends on different regulatory elements within the transcript and this makes a possible application significantly difficult.