Difference between revisions of "Team:MIT/3mk-HBG"
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| − | <p>After designing our 2-exon mKate HBG reporter, which only showed mKate knockdown, we needed a positive reporter. We designed two 3-exon reporters, a split mKate reporter and a dual fluorescence reporter | + | <p>After designing our 2-exon mKate HBG reporter, which only showed mKate knockdown, we needed a positive reporter. We designed two 3-exon reporters, a split mKate reporter and a dual fluorescence reporter. </p> |
<h3> 3-exon mKate Fluorescent Reporter </h3> | <h3> 3-exon mKate Fluorescent Reporter </h3> | ||
Revision as of 21:03, 29 October 2017
Experiments with 3 Exon mKate HBG Reporter
After designing our 2-exon mKate HBG reporter, which only showed mKate knockdown, we needed a positive reporter. We designed two 3-exon reporters, a split mKate reporter and a dual fluorescence reporter.
3-exon mKate Fluorescent Reporter
The reporter was designed to contain mKate exon one, HBG Intron two, the REST-4 included exon from our disease model, HBG Intron one, and mKate exon two.
The mKate exons and the HBG Intron two were taken from the 2-exon mKate reporter. The REST-4 exon was chosen because it contains a stop codon, and because we wanted our reporter to mirror our disease model. The HBG Intron one was chosen based on the literature. [Source]. A g-block was designed with the REST exon, HBG intron 2 and complementary sticky ends to be added to the 2-exon mKate HBG reporter between HBG Intron 2 and mKate exon two.
If our guide system is not present in the cell, mKate exon one, the REST exon, and mKate exon two will be spliced together. During translation, the stop codon in the REST exon will cause the ribosome to fall off, resulting in an incomplete mKate and therefore a knockdown of red fluorescence.
When our guide system is introduced to the cells, it will cover the 5’ splice site on HBG Intron 2, causing the spliceosome to skip over the REST exon. The cell will then be able to translate mKate as normal, and we expect to see a rise in red fluorescence. This reporter therefore achieves our goal of being a positive reporter.
