Difference between revisions of "Team:Calgary/Notebook"
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| − | + | We began an extensive literature search on the topic of insulators and other epigenetic regulatory elements, such as scaffolding/matrix attachment regions (S/MARs) and ubiquitous chromatin remodeling elements (UCOEs). We determined that UCOEs will be most effective at opening chromatin and will be very useful in producing continuous high-level transcription of any transgenes that are inserted into the human cell genome via our CRISPR/FLP system. While UCOEs will allow for our gene to enter an active transcriptional state, insulators will function to maintain this state. If flanking the transgene, insulators protect the gene from the spread of neighboring heterochromatic regions and from interaction with nearby enhancers. In addition, we began a discussion about the testing platform that we will utilize. Working in parallel with the CRISPR subgroup prevents us from using their integration scheme, so we are investigating other options for preliminary experiments. We also completed all necessary Lab Safety training which will allow us to work in the lab this summer. | |
</p> | </p> | ||
<br> | <br> | ||
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| − | Week 2 | + | Week 2 (7th to 13th) |
</h5> | </h5> | ||
<p> | <p> | ||
| − | + | We continued with extensive literature research specifically focused on ubiquitous chromatin remodeling elements (UCOEs) as well as insulators. The A2UCOE, CBX3-UCOE, and cSH4 insulator were selected for experimentation and their sequences were obtained. We began planning constructs which will eventually constitute the various cassettes during recombinase mediated cassette exchange (RMCE) procedures. We continued the investigation of other options for preliminary experiments that will free us from dependence on the CRISPR subgroup's FRT integration scheme. One promising approach would be to generate minicircles from triple-FRT containing FLP plasmids within prokaryotic cells, isolate and purify the minicircles, and co-transfect them with plasmids expressing FlpO (pCAG-FLPo) into a HEK cell line that already contain one FRT site, such as the Flp-In T-REx HEK293 cells available commercially. This would allow for the insertion of a second heterospecific FRT site, which makes RMCE possible. Therefore, we could conduct our experiments on UCOEs and insulators while the CRISPR subgroup fine-tunes the CRISPR-mediated FRT integration scheme. We began a discussion about the process of obtaining the Flp-In T-REx HEK293 cells from internal contacts within the University of Calgary. Another promising possibility explored is to obtain a cell line that already contains heterospecific FRT sites. In this case, we could begin immediately with RMCE in order to investigate UCOE and insulator function. We contacted the principle investigator from that research group in regards to obtaining cells from the generated cell line and are awaiting a response. | |
</p> | </p> | ||
<br> | <br> | ||
Revision as of 23:51, 14 October 2018
NOTEBOOK
Below are notes regarding all CRISPR experiments.
May
Week 1
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas vitae feugiat odio. Morbi luctus lacus in suscipit ultrices. Suspendisse quis ultricies sapien. Vestibulum vitae massa eu dolor cursus aliquam vitae eget neque. Mauris a turpis nec mauris luctus blandit. Nunc suscipit, nisl nec fringilla placerat, magna nisi imperdiet est, nec interdum quam nulla nec quam. Nunc ligula dolor, convallis quis placerat eget, dapibus ut diam. In quis iaculis magna, a tristique magna.
Week 2
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas vitae feugiat odio. Morbi luctus lacus in suscipit ultrices. Suspendisse quis ultricies sapien. Vestibulum vitae massa eu dolor cursus aliquam vitae eget neque. Mauris a turpis nec mauris luctus blandit. Nunc suscipit, nisl nec fringilla placerat, magna nisi imperdiet est, nec interdum quam nulla nec quam. Nunc ligula dolor, convallis quis placerat eget, dapibus ut diam. In quis iaculis magna, a tristique magna.
Below are notes regarding all CME (chromatin modifying elements) experiments.
May
Week 1 (1st to 6th)
We began an extensive literature search on the topic of insulators and other epigenetic regulatory elements, such as scaffolding/matrix attachment regions (S/MARs) and ubiquitous chromatin remodeling elements (UCOEs). We determined that UCOEs will be most effective at opening chromatin and will be very useful in producing continuous high-level transcription of any transgenes that are inserted into the human cell genome via our CRISPR/FLP system. While UCOEs will allow for our gene to enter an active transcriptional state, insulators will function to maintain this state. If flanking the transgene, insulators protect the gene from the spread of neighboring heterochromatic regions and from interaction with nearby enhancers. In addition, we began a discussion about the testing platform that we will utilize. Working in parallel with the CRISPR subgroup prevents us from using their integration scheme, so we are investigating other options for preliminary experiments. We also completed all necessary Lab Safety training which will allow us to work in the lab this summer.
Week 2 (7th to 13th)
We continued with extensive literature research specifically focused on ubiquitous chromatin remodeling elements (UCOEs) as well as insulators. The A2UCOE, CBX3-UCOE, and cSH4 insulator were selected for experimentation and their sequences were obtained. We began planning constructs which will eventually constitute the various cassettes during recombinase mediated cassette exchange (RMCE) procedures. We continued the investigation of other options for preliminary experiments that will free us from dependence on the CRISPR subgroup's FRT integration scheme. One promising approach would be to generate minicircles from triple-FRT containing FLP plasmids within prokaryotic cells, isolate and purify the minicircles, and co-transfect them with plasmids expressing FlpO (pCAG-FLPo) into a HEK cell line that already contain one FRT site, such as the Flp-In T-REx HEK293 cells available commercially. This would allow for the insertion of a second heterospecific FRT site, which makes RMCE possible. Therefore, we could conduct our experiments on UCOEs and insulators while the CRISPR subgroup fine-tunes the CRISPR-mediated FRT integration scheme. We began a discussion about the process of obtaining the Flp-In T-REx HEK293 cells from internal contacts within the University of Calgary. Another promising possibility explored is to obtain a cell line that already contains heterospecific FRT sites. In this case, we could begin immediately with RMCE in order to investigate UCOE and insulator function. We contacted the principle investigator from that research group in regards to obtaining cells from the generated cell line and are awaiting a response.
Below are notes regarding all dry lab activities.
May
Week 1
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas vitae feugiat odio. Morbi luctus lacus in suscipit ultrices. Suspendisse quis ultricies sapien. Vestibulum vitae massa eu dolor cursus aliquam vitae eget neque. Mauris a turpis nec mauris luctus blandit. Nunc suscipit, nisl nec fringilla placerat, magna nisi imperdiet est, nec interdum quam nulla nec quam. Nunc ligula dolor, convallis quis placerat eget, dapibus ut diam. In quis iaculis magna, a tristique magna.
Week 2
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas vitae feugiat odio. Morbi luctus lacus in suscipit ultrices. Suspendisse quis ultricies sapien. Vestibulum vitae massa eu dolor cursus aliquam vitae eget neque. Mauris a turpis nec mauris luctus blandit. Nunc suscipit, nisl nec fringilla placerat, magna nisi imperdiet est, nec interdum quam nulla nec quam. Nunc ligula dolor, convallis quis placerat eget, dapibus ut diam. In quis iaculis magna, a tristique magna.