In response to cardiac cell death from an injury, zebrafish, as opposed to mammals, are able to regenerate new heart cells without significant scar tissue. Heart attacks, a leading cause of death in the United States, leave behind substantial scar tissue that weakens the heart and leads to a greater chance of a repeated cardiac event. Many genes and major molecular pathways are highly conserved from fish all the way to humans; thus, understanding how the regenerative process works in zebrafish may provide insight into potential therapies for heart attacks in humans. However, we must first understand how heart regeneration occurs in zebrafish at the molecular level. From the time of injury to a zebrafish heart through the completion of regeneration, we want to build a regulatory network showing which genes are up- or down-regulated and how they are interconnected.
Transcription factors, such as tcf21 and tbx20, bind to regulatory elements of DNA and can either upregulate or downregulate nearby genes. To build this gene regulatory network, scientists use a technique called ChIP-seq that can determine where in the genome these transcription factors bind. Nearby genes are potential targets of their regulation, and we can validate these enhancers by testing differences in expression using a fluorescent protein reporter construct.
ChIP-seq requires high quality antibodies capable of specifically recognizing the transcription factor of interest. These are rarely available. Because each different antibody that is used requires validation and optimization for ChIP-seq, it is not easy to scale up the collection of data for different transcription factors. One way to get around these problems is to express a tagged version of the transcription factor. The tag is recognizable by the same antibody; however, expressing the tagged transcription factor in this manner almost inevitably results in higher than normal levels of expression, leading to false positives in the ChIP-seq data.
Using CRISPR/Cas9 technology to target and modify specific sequences in the genome, we developed a novel method to add an epitope tag to these transcription factors at their endogenous loci. This allows us to run ChIP-seq experiments with the transcription factor at physiological levels of expression. We can also use the same antibody to eliminate repeated validation and optimization steps.
We have successfully tagged two genes that may be involved in heart regeneration, tcf21 and tbx20. tcf21 is expressed in the developing epicardium and is required for the proper development of the branchial arches. tbx20 is expressed in the cardiomyocytes and is required for the proper development of the heart, and it has also been shown to be upregulated in response to injury in the zebrafish. With tbx20, we have performed a successful ChIP-seq experiment and have tested several promising target genes.
It is difficult to test if either tcf21 or tbx20 is required for regeneration, as both of these genes are essential for development and mutants do not survive. The solution to this problem is to engineer the gene so that it can be turned off at a specific time and in a specific cell type. A common method of this in the mouse model utilizes the Cre/loxP system: two loxP sites flank a required segment of a gene, and the introduction of the enzyme Cre deletes the DNA between them. Until CRISPRs this was not feasible in zebrafish, which lacked an efficient method of targeted modification in the genome. We adapted our method for integrating epitope tags to add the two loxP sites in the genome. We have made and tested a fully conditional mutant for tbx20, and we have put in the first of the two loxP sites for tcf21. / Biology
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/303 |
| Date | January 2020 |
| Creators | Burg, Leonard |
| Contributors | Balciunas, Darius, Habas, Raymond, Amini, Shohreh, Clark, Karl |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 94 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/287, Theses and Dissertations |
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