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Engineering recombinant antibodies for the detection of axonal FMRP spliceforms
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Engineering recombinant antibodies for the detection of axonal FMRP spliceforms

Steven Gardner Doll
Master of Science (M.S.), Drexel University
Jan 2016
DOI:
https://doi.org/10.17918/etd-6693
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Abstract

Biology Epigenetics--Research--Methodology Fragile X syndrome X-linked mental retardation Neuroplasticity
Epigenetic silencing of the fragile X mental retardation protein (FMRP) is understood to be the primary causative agent of fragile X syndrome (FXS), a well characterized cause of intellectual disability and autism. This raises the question as to the functions of FMRP in the normal brain, and how loss of the protein results in dysregulated plasticity. FMRP is an mRNA binding protein that negatively regulates translation of its mRNA targets. Attempts to identify mRNA sequence motifs and mRNA secondary structures that FMRP might recognize has only managed to account for a fraction of the total mRNAs the protein may associate with. Furthermore, mRNAs are large, and would likely require the effects of multiple proteins to stall translation. Thus, it may be the case that FMRP operates alongside other proteins in a co-regulatory complex. In order to probe the functions of FMRP, it is necessary to have the proper tools and a working model. To the first point, a method for the production of recombinant antibodies against FMRP has been devised that makes use of simple molecular techniques including PCR and restriction cloning as opposed to maintenance of anti-FMRP producing hybridomas. To the second point, we have tested a mouse model by which the olfactory bulb may be used to detect specific isoforms of FMRP. Localization of specific isoforms to a discrete region of the neuron in vivo would suggest a particular function of those isoforms. If FMRP does not operate alone, this would suggest that specific isoforms are required to participate in specific protein complexes. Loss of FMRP expression would disrupt formation of these complexes and their effects upon translational regulation. We were able to successfully produce a method for the cloning of recombinant anti-FMRP antibody. However, the mouse model through which we would detect specific FMRP isoforms proved incapable of providing interpretable results. Nonetheless, we wish to test our method of recombinant antibody production in an effort to provide a useful technique for the community on the whole and eliminate the need to maintain anti-FMRP hybridoma.

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