Date of Award

8-2014

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Carlos A. Molina

Committee Member

Sandra D. Adams

Committee Member

Kirsten J Monsen

Abstract

Genome editing has become an important tool in identifying the specific function and role of a gene in an organism. With the advent of genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems, editing a gene has become much easier and less expensive. This CRISPR/Cas9 system uses a small 20 nucleotide long guided RNA (gRNA), which along with Cas9 will bind to the target site and cleave it. In this research, CRISPR/Cas9 system was used to knock out the inducible cAMP early repressor (ICER) promoter sequence in zebra fish. ICER has anti-proliferative activity and acts as a tumor suppressor. In Ras-induced melanoma, ICER protein is being targeted to degradation. Knocking out the ICER gene will help us to establish the tumorigenicity of ICER in melanomas.

A gRNA, specific towards the ICER promoter sequence was designed in an attempt to cleave it with Cas9. Plasmids pDR274(-)atgICER and pMLM3613 were used to generate gRNA and Cas9mRNA via in vitro transcriptions. To check the efficiency of designed gRNA in vitro, PAC-2 cell lines were transfected with a plasmid that expressed both gRNA and Cas9. The results demonstrate the ability of Cas9 to cleave the target sequence. In the future, this plasmid could be used to perform microinjection in zebra fish embryos with the generated gRNA and Cas9mRNA.

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