Read the following paper and answer the questions below: Tillotson, R., Selfridg

Question

Read the following paper and answer the questions below:

Tillotson, R., Selfridge, J., Koerner, M.V., Gadalla, K.K., Guy, J., De Sousa, D., Hector, R.D., Cobb, S.R. and Bird, A., 2017. Radically truncated MeCP2 rescues Rett syndrome-like neurological defects. Nature, 550(7676), p.398.

Please initially read the paper to follow the logic and the general concepts – do not become “bogged down” in the minutiae. The goal is to understand the paper sufficiently (i.e. conceptually) in order to address the following questions and not to become an expert on every technique. Nonetheless, one should briefly look up any techniques that seem relevant and are required to enable you to move forward in the paper.

1. What is the overall hypothesis and why is this important for Rett syndrome? What is the experimental strategy to test this hypothesis?

2. Tillotson et al. (2017) used a floxed STOP cassette to regulate expression of their NIC transgene. What was unusual (and why) about the lifespan of the control STOP mice lacking the creERT transgene (when compared to MeCP2 null mice) and how is it possible to account for this phenotype?

3. Discuss any caveats associated with using male mice as the experimental model for Rett syndrome. Given that male mice with internal testes are infertile why aren’t MeCP2-/- female mice routinely used as a model of Rett syndrome?

4. How has the Tsix gene been used recently to assess the role of MeCP2 in female mouse models of Rett syndrome?

Explanation / Answer

1. Here, the overall hypothesis is, in mice a X-linked MECP2 gene physically interacts with DNA via it’s NCoR/SMRT complex. Any mutation in this complex cause Rett syndrome (RTT).

The experimental strategy used was almost all amino acid sequences were removed except the Mrthyl-CpG Binding Domain (MBD) and NCoR/SMRT Interaction Domain (NID). These domains are sufficient to avoid RTT like defects. They generated delN and delNC knock-in-mice by replacing the endogenous Mecp2 alleles in ES cells, which were used to produce germline-transmitting chimaeras. Truncated proteins were exposed at approximately WT levels in brain and in neurons. To check phenotypes, knock-in-mice were crossed onto a background strain and cohorts underwent weekly phenotypic behavioral tests.

2. Tillotson et al. (2017) used a floxed STOP cassette to regulate expression of their NIC transgene. It was unusual about the lifespan of the control STOP mice lacking the creERT transgene (when compared to MeCP2 null mice) that control mice developed severe symptoms and failed to survive beyond 26 weeks.

3. Male mice have XY as their sex chromosome. We found that RTT is a disease associated with a heterozygous mutation in MECP2 gene which is X-linked. So, if the mutation is found in one allele in the X chromosome, it will generate the disorder, as male has only one X chromosome. So, it will be easy to test.

Though, male mice have their infertile internal testes, MeCP2-/- female mice are not used as a model of Rett syndrome, because, syndrome occur only in case of heterozygous condition.

4. Currently, there are no treatment for RTT, but MECP2 restoration through reactivation of the inactive X (Xi) has been of considerable interest. Progress of the Xi-reactivation therapy has been hampered by a lack of suitable female mouse models. Because of cellular mosaicism due to random X-chromosome inactivation (XCI), Mecp2+/- heterozygous females develop only mild RTT. Recently, a mouse model has been created by introducing a mutation in Tsix, the antisense regulator of XCI allelic choice. Tsix-Mecp2 mice may show reduced MECP2 mosaicism and almost closely phenocopy the severely affected Mecp2-null males.

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