Artificial Intron Technology To Generate Conditional Knock-Out Mice

Author

Amber N. Thomas-Gordon, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical SciencesFollow

Author ORCID Identifier

0000-0002-1319-5789

Date of Graduation

8-2020

Document Type

Thesis (MS)

Program Affiliation

Biomedical Sciences

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Jan Parker-Thornburg, Ph.D.

Committee Member

Richard Behringer, Ph.D.

Committee Member

Carrie Cameron, Ph.D.

Committee Member

Vicki Huff, Ph.D.

Committee Member

Jason Heaney, Ph.D.

Abstract

Genetic engineering has been re-shaped by the invention of new tools in modern biotechnology in a way that offers precision and efficiency in modifying the genome at a single nucleotide level and/or allowing precise control of gene expression. Such gene manipulation brings about significant findings and revelations in comprehending more about embryonic development, cellular and physiological functions, and disease pathology. Current methods used to produce conditional knockouts have limitations on conditional allele placement and modification varies among genes in different organisms. Thus, a system for generating conditional alleles with fidelity remains a challenge. My goal was to examine an approach for generating conditional inactivation of targeted genes that uses a small standardized intron that should be easily introduced using CRISPR/Cas9 methodology. I tested the DECAI (DEgradation based on Cre-regulated- Artificial Intron) artificial intron technology, designed by Dr. Tilmann Burckstummer of Horizon Discovery Ltd., in Vienna Austria, in mouse embryonic stem cells (mESC). Given similarities of the splicing machinery between human and mouse, I expected that the artificial intron components would successfully produce conditional alleles of mouse genes. This intron is designed with loxP sites flanking the branch point sequence, removal of which will abrogate splicing and expose translational stop sequences. This small cassette was inserted into the 5’ portion of the coding region of the Neomycin resistance gene (Neo^R) and this construct was randomly integrated into mouse ES cells. To test its utility for gene inactivation, a Hygromycin resistance plasmid containing Cre- recombinase was introduced into the intron-containing mESCs. A plus/minus screen for Neomycin resistance was performed and Sanger based sequencing was done to identify whether a conditional knock-out had been generated. My results show that the Neo^R gene and the artificial intron remained intact and conditional gene inactivation did not occur. However, with thorough investigation of the gene of interest and splicing requirements, this artificial intron technology has potential for practical use in various model systems.

Keywords

artificial intron, conditional knock-outs, knock-out mice, small cassette, introns, splicing, conditional alleles, genetic engineering, mouse embryonic stem cells, Neomycin resistance gene