Title

Modeling 3D Retinogenesis in Mouse Embryonic Stem Cells Following CRISPR-mediated Crx Knockdown

Graduation Date

5-2017

Document Type

Master's Thesis

Degree Name

Master of Science

Department or Program

Biological Sciences

Department or Program Chair

Maggie Louie, Ph.D.

First Reader

Deepak Lamba, Ph.D.

Second Reader

Mary Sevigny, Ph.D.

Abstract

An emerging technology known as three-dimensional (3D) tissue engineering has allowed scientists to mimic tissues found in vivo. Previous studies indicate that it is possible to differentiate dissociated mouse embryonic stem cells (mESCs) into 3D retinal tissues in vitro (Bertacchi, 2015; Eiraku, 2012). The newly differentiated retinal tissues are said to encompass all of the major components found in retinal tissues. The generation of in vitro 3D tissues holds great potential in terms of patient-specific disease modeling. Although various diseases have been well-studied in animal models, there are limitations with regards to patient-specificity. The generation of animal models to study each specific mutation would be impractical and costly. With in vitro generation of tissues and organs, scientists have the capacity to generate disease models consisting of mutations specific to patients.

There are various gene editing techniques available to generate and recreate disease-specific 3D disease models using stem cells. The newest gene editing technique known as Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) has allowed scientists to edit the genome with greater precision than other genome editing technologies like Zinc Finger Nucleases (ZFNs) or Transcription Activator-like Effector Nucleases (TALENs).

In order to overcome current limitations on disease-specific model systems, we developed a protocol for differentiating mouse embryonic stem cells into 3D retinas in vitro. In addition, to confirm the utility of this 3D model for future disease-specific mutations, we utilized the CRISPR method as a means to knockdown the transcription factor CRX, which is known to regulate key developmental decisions during retinogenesis, and as such, mutations in CRX have resulted in various diseases such as Leber Congenital Amaurosis, Cone-Rod Dystrophy, and Retinitis Pigmentosa.

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