The Effect of Creating Gene Specific Knockout Zebrafish on Expression Patterns Using CRISPR-Cas-9 System
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Cancer takes several thousands of lives every year worldwide, yielding a lot of research regarding the impacts of specific genes in the development of cancer metastasis. This specific study looks at the expression of Cancer-Testis (C/T) genes with regards to activation and deactivation of specific long non-coding RNA (lnc RNA). One specific long non-coding RNA, HiClinc-1, has shown high expression in embryogenesis and reexpression in cancer tissue in the testis. It has been determined that this specific long noncoding RNA is a Cancer-Testis long non-coding RNA (C/T lnc RNA). An RNA pull down and Mass spectrophotometry was conducted and it was determined that HiClinc-1 binds to two binding proteins called Insulin-like growth factor-II-mRNA-binding protein 1 and 3 (igf2bp1, igf2bp3). This study looked at the expression patterns of the Insulin-like growth factor-IImRNA- binding protein 1 and 3 and found that they were nearly identical to that of HiClinc-1’s expression patterns. A knockout of both Insulin-like growth factor-II-mRNAbinding protein 1 and 3 in zebrafish embryo was created to compare the defects of the zebrafish knockouts to those of the HiClinc-1 knockout zebrafish. The method used in this process was using clustered regular inter short palindromic repeats and associated nucleases (CRISPR-cas9 protocol). It was determined that the insertion of the guide RNA from the CRISPR-cas9 system successfully created mutations in the zebrafish embryo and more data will be collected in the future. Mutations which are expected are three types of zebrafish- wildtype, homo and hetero. If phenotypes in the knockout zebrafish replicate that of the HiClinc-1 knockouts, it could imply that HiClinc-1 functions by binding to igf2bp1 and igf2bp3.